Chimera comprising bacterial cytotoxin and methods of using the same

ABSTRACT

This invention provides, a recombinant polypeptide encoding a chimera. The chimera includes a DNase I fragment or a homologue thereof and a Cdt fragment or a homologue thereof. Further, the invention provides methods, utilizing the recombinant polypeptide encoding the chimera, such as a method for inhibiting the proliferation of a neoplastic cell, a method for treating a neoplastic disease in a human subject, a method for inhibiting or suppressing a neoplastic disease in a human subject, and a method for reducing the symptoms associated with a neoplastic disease in a human subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/858,312, filed Aug. 17, 2010, which is a continuation-in-part of PCTPatent Application PCT/US2009/038740, filed Mar. 30, 2009, which claimspriority to U.S. Provisional Patent Application 61/064,862, filed Mar.31, 2008, all of which are incorporated by reference herein in theirentirety.

GOVERNMENT INTEREST STATEMENT

This invention was made with government support under grant no. R21DE017679 awarded by the National Institute of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to recombinant polypeptides comprising a chimerathat inhibits cell proliferation and a method of using the same.

BACKGROUND OF THE INVENTION

Over the last few decades there has been developing interest innon-traditional treatments for certain types of cancer. Recombinanttoxins, hybrid proteins composed of a bacterial toxin and either agrowth factor or a portion of a recombinant monoclonal antibody, havereceived significant attention in cancer therapeutics.

Human DNase I has been used as a therapeutic agent for the treatment ofwounds and ulcers, bronchitis, inflammatory conditions, herpes infectionand most notably, cystic fibrosis. The cytolethal distending toxin (Cdt)is a genotoxin, produced by several species of bacteria including theperiodontal pathogen Actinobacillus actinomycetemcomitans.

Cytolethal distending toxin (Cdt) is a family of secreted bacterialprotein holotoxins that classically arrest the growth of specific typesof eukaryotic cells or cell lines at either the G0/G1 or G2/M phase ofthe cell cycle. The holotoxin is the product of three genes expressed bya handful of facultative or microaerophilic gram-negative pathogenicbacterial species. The species identified to date that express abiologically active CDT include select strains of enteropathogenicEscherichia coli, Campylobacter jejuni, Campylobacter upsaliensis,Campylobacter coli, Shigella dysenteriae, Haemophilus ducreyi,Helicobacter hepaticus, Helicobacter flexispira, Helicobacter bilis,Helicobacter canis and, the periodontal pathogen, Actinobacillusactinomycetemcomitans. Organization of the genetic locus as well as thestructure and biological activity of the holotoxin are fairly wellconserved among the bacterial genera that express the Cdt. Biologicallyactive toxin is a heterotrimer composed of approximately 18-25 kDa(CdtA), 31 kDa (CdtB) and 21 kDa (CdtC) protein subunits expressed froma polycistronic operon. An adjunct property of the cdt genes is thatthey appear to have a eukaryotic rather than prokaryotic heritage. Thecdt gene products exhibit deduced amino acid sequence andstructure/function similarities (albeit weak) to those of eukaryoticproteins.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, a recombinant polypeptidecomprising a chimera, wherein said chimera comprises a DNase I fragmentor a homologue thereof and a Cdt fragment or a homologue thereof.

In another embodiment, the present invention provides a recombinantpolynucleotide encoding a recombinant polypeptide comprising a chimera,wherein said chimera comprises a DNase I fragment or a homologue thereofand a Cdt fragment or a homologue thereof.

In another embodiment, the present invention provides a DNA vectorcomprising a recombinant polynucleotide encoding a recombinantpolypeptide comprising a chimera, wherein said chimera comprises a DNaseI fragment or a homologue thereof and a Cdt fragment or a homologuethereof.

In another embodiment, the present invention provides a plasmidcomprising a recombinant polynucleotide encoding a recombinantpolypeptide comprising a chimera, wherein said chimera comprises a DNaseI fragment or a homologue thereof and a Cdt fragment or a homologuethereof.

In another embodiment, the present invention provides a method forinhibiting the proliferation of a neoplastic cell comprising the step ofcontacting said cell with a recombinant polypeptide comprising a chimeraor a nucleic acid molecule encoding the same, wherein said chimeracomprises a DNase I fragment or a homologue thereof and a Cdt fragmentor a homologue thereof, thereby inhibiting the proliferation of aneoplastic cell.

In another embodiment, the present invention provides a method fortreating a neoplastic disease in a subject comprising the step ofadministering to said subject a recombinant polypeptide comprising achimera or a nucleic acid encoding the same, wherein said chimeracomprises a DNase I fragment or a homologue thereof and a Cdt fragmentor a homologue thereof, thereby treating a neoplastic disease in asubject.

In another embodiment, the present invention provides a method forinhibiting or suppressing a neoplastic disease in a subject comprisingthe step of administering to said subject a recombinant polypeptidecomprising a chimera or a nucleic acid encoding the same, wherein saidchimera comprises a DNase I fragment or a homologue thereof and a Cdtfragment or a homologue thereof, thereby inhibiting or suppressing aneoplastic disease in a subject.

In another embodiment, the present invention provides a method forreducing the symptoms associated with a neoplastic disease in a subjectcomprising the step of administering to said subject a recombinantpolypeptide comprising a chimera or a nucleic acid encoding the same,wherein said chimera comprises a DNase I fragment or a homologue thereofand a Cdt fragment or a homologue thereof, thereby reducing the symptomsassociated with a neoplastic disease in a subject.

In another embodiment, the invention provides a composition comprising:a recombinant CdtA polypeptide comprising at least one of the mutationsC149A and C178A, said polypeptide operably linked to a ligand that bindsspecifically to an antigen expressed on the surface of a cancerousepithelial cell type.

In another embodiment, the invention provides a recombinant CdtApolypeptide operably linked to a ligand that binds specifically to anantigen expressed on the surface of a cancerous epithelial cell type,wherein said recombinant CdtA polypeptide comprises at least one of themutations C149A and C178A.

In another embodiment, the invention provides a chimeric CdtApolypeptide comprising at least one of the mutations C149A and C178A.

In another embodiment, the invention provides an isolated nucleic acidsequence encoding (i) a CdtA polypeptide comprising at least one of themutations C149A and C178A or (ii) a nucleic acid sequence that is atleast 85% identical to the sequence of (i).

In another embodiment, the invention provides a method for inhibitingthe proliferation of a cancerous epithelial cell type comprising:administering a recombinant CdtA polypeptide comprising at least one ofthe mutations C149A and C178A, said polypeptide operably linked to aligand that binds specifically to an antigen expressed on the surface ofa cancerous epithelial cell type.

In another embodiment, the invention provides a method for inhibitingthe proliferation of a cancerous epithelial cell comprising: contactingsaid cell with a recombinant CdtA polypeptide comprising at least one ofthe mutations C149A and C178A, said polypeptide operably linked to aligand that binds specifically to an antigen expressed on the surface ofsaid cell.

In another embodiment, the invention provides a method for treatingcancer comprising: administering a recombinant CdtA polypeptidecomprising at least one of the mutations C149A and C178A, saidpolypeptide operably linked to a ligand that binds specifically to anantigen expressed on the surface of a cancerous epithelial cell type.

In another embodiment, the invention provides a method for treating adisease associated with oral candidiasis comprising: administering arecombinant CdtA polypeptide comprising at least one of the mutationsC149A and C178A, said polypeptide operably linked to a ligand that bindsspecifically to an antigen expressed on the surface of a cancerousepithelial cell type.

In another embodiment, the invention provides a method for treating adisease associated with oral candidiasis comprising: administering atoxin composition specific to Candida albicans, said toxin compositioncomprising a recombinant CdtA polypeptide comprising at least one of themutations C149A and C178A, said polypeptide operably linked to a ligandthat binds specifically to an antigen expressed on the surface of acancerous epithelial cell type.

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Characterization of recombinant CdtB-His₆. (A) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of affinity purifiedrecombinant A. actinomycetemcomitans CdtB-His₆. The gel is stained withCoomassie Brilliant Blue. (B) Dose response kinetics of the conversionof supercoiled (S) plasmid DNA to relaxed (R) and linear (L) forms byCdtB-His₆. Agarose gel stained with ethidium bromide. The activity ofCdtB labeled with a histidine tag at the amino terminal end of theprotein is compared at the highest concentration (1 μg/1 μg of DNA) ofCdtB-His₆ tested. (C) Dose response kinetics of the activity of bovineDNase I.

FIG. 2. DNA nicking assay containing increasing concentrations of eitherMgCl₂ (FIG. 2A; top panel), CaCl₂ (FIG. 2B; middle panel) or MnCl₂ (FIG.2C; bottom panel) in the standard reaction buffer. Supercoiled (S),relaxed (R) and linear (L) forms of DNA were quantified by densitometryof ethidium bromide stained agarose gels (insets). The data shown isrepresentative of several repeat experiments

FIG. 3. Heat liability of recombinant CdtB-His₆ and DNase I. (FIG. 3A)CdtB-His₆ heated for various periods in boiling water and then assayedfor supercoiled DNA nicking activity. (FIG. 3B) bovine DNase I treatedas in panel A. (FIG. 3C) effects of heating on the nicking activity ofCdtB-DNase I and DNase I-CdtB. Numbers above gels lanes representminutes. Abbreviations are the same as in FIG. 1.

FIG. 4. The effect of globular (G) actin on the supercoiled DNAs nickingactivity of CdtB-His₆, bovine DNase I and the two hybrid proteins.Abbreviations are the same as in FIG. 1.

FIG. 5. (FIG. 5A) Computer models of the A. actinomycetemcomitans CdtB(as part of the Cdt heterotrimer) and bovine DNase I. (FIG. 5B)Theoretical model of the CdtB/DNase I hybrid protein construct. The CdtB(amino-terminal half) and DNase I (carboxy-terminal half) portions ofthe hybrid protein are shown in orange and magenta, respectively. Theamino-(N) and carboxy (C)-termini are labeled. (FIG. 5C) Sequencealignment of the four His-tagged constructs (CdtB-H (SEQ ID NO: 16);CdtB/DNaseI (SEQ ID NO: 26); DNaseI/CdtB (SEQ ID NO: 27); human DNAse I(SEQ ID NO: 7)). Conserved and identical residues are marked by dots andasterisks, respectively. The blue and orange arrows mark the gene fusionposition and predicted signal sequence cleavage sites, respectivelyAmino acid substitutions are marked in green and predicted functionaldomains are in red. Location of the nuclear localization signal (NLS)and information about the location of functional residues in DNase I wasfrom Nishikubo et al. (2003) and Pan et al. (1998), respectively.Disulfides in DNase I are designated by brackets.

FIG. 6. The biological and binding activities of the hybrid proteins.(FIG. 6A) heterotoxins were reconstituted with wild-type CdtA and CdtCand each of the hybrid proteins. CHO cells were treated with thepreparations. Colonies were fixed, stained and counted after growth forsix days and expressed as colony-forming units (CFU). (FIG. 6B)Saturation binding kinetics of the two hybrid proteins were compared tothat of CdtB-His₆. The same proteins were incubated with wild-type CdtAand CdtC in refolding buffer and subjected to differential dialysis. Thereconstituted samples were examined before (BD) and after (AD) dialysison a western blot (inset). (FIG. 6C) The stoichiometric binding of thetwo hybrid proteins to wild-type CdtA and CdtC immobilized onthyroglobulin was examined by ELISA. Numbers above the columns areabsorbance ratios calculated as described in Methods. The hybridproteins, and CdtB-His₆ as a control, were mixed with wild-type CdtA andCdtC, as in the differential dialysis experiment in the inset in panelA, and the reconstituted samples assayed for DNA nicking activity(inset). The reaction examined in the first lane contained non-complexedCdtB-His₆. Abbreviations are the same as in the legend for FIG. 1.Experiments were performed a minimum of three times. Mean values andstandard deviations were plotted where appropriate.

FIG. 7. A computer models of CdtB (as part of the Cdt heterotrimer) andthe two hybrid proteins. The α-helix H1 and large loop domains arelabeled. Note that the orientation of the DNase I/CdtB structure isflipped 180° relative to that of CdtB/DNase I. The A.actinomycetemcomitans Cdt was modeled using the program UCSF Chimeraversion 1.2197 and PDB coordinate file 2F2F. The theoretical hybridprotein structures were generated with Modeller 9.1 and visualized withUCSF Chimera.

FIG. 8. Nuclease activity of CdtB/DNase I^(mut5) was compared to that ofCdtB-His₆ and the original CdtB/DNase I construct. The data is expressedas a percentage of the supercoiled (S) form DNA remaining after thereaction. These values were obtained by densitometry of ethidium bromidestained agarose gels (inset A). Semilogarithmic plot of the log of thesubstrate concentration [S] versus time of the reaction (expressed inminutes) for CdtB/DNase I^(mut5) (inset B). [S] represents the amount ofS-form DNA obtained by densitometry of the agarose gel in the inset).The control lacked enzyme. The data shown is representative of severalrepeat experiments.

FIG. 9 depicts a flow cytometry of untreated and Cdt-treated HeLa cells,the human cancer cell line CAL-27 and primary HGEC (AL-1). Culturestreated with Cdt received 10 μg of reconstituted recombinant protein/ml.Cell nuclei were prepared 36 h post-intoxication and were stained withpropidium iodide. Quantitative values for the diploid G1, diploid G2 andS peaks are provided in Table 4.

FIG. 10 shows computer models of CdtB-His₆ and CdtB/DNase I^(mut5)containing the large loop substitution. The amino- and carboxy-terminalhalves of the proteins are in red and magenta, respectively. The α-helixH1 and large loop domains (yellow) are labeled. Distances are labeled inangstroms. The theoretical computer models were generated as describedin the legend to FIG. 6.

FIG. 11 is a bar graph showing wild-type and hybrid proteins that wereadded sequentially to CdtA pre-bound to thyroglobulin-coated 96-wellplates. Bound protein was detected as described in the legend to FIG.6C.

FIG. 12 is a bar graph showing heterotoxins that were reconstituted withwild-type CdtA, wild-type CdtC and either wild-type CdtB or CdtB/DNaseIY174P (SEQ. ID NO: 14 and 15). CHO cells were treated with thepreparations. Colonies were fixed, stained and counted after growth forsix days and expressed as colony-forming units (CFU).

FIG. 13 depicts flow cytometry of HeLa, CCL-30 and CAL-27 cancer celllines with heterotoxin containing CdtB or CdtB/DNase I^(Y174P). Culturestreated with heterotoxin received 10 μg of reconstituted recombinantprotein/ml. Cell nuclei were prepared 36 h post-intoxication and werestained with propidium iodide. Quantitative values for the diploid G1,diploid G2 and S peaks are provided in Table 5.

FIG. 14 is a phylogenetic tree showing the relationship of bacterialCdtB and mammalian DNase I deduced amino acid sequences. GenBankaccession numbers are given in parentheses.

FIG. 15. Cell cycle arrest of epithelial cells following exposure to theCdt. Cultures (24 h) of primary HGEC, CAL-27 (SCC) and HeLa cells wereleft untreated or treated with 10 μg/ml of reconstituted toxin(CdtABC-treated)/3.5×10⁶ cells for 36 h. Propidium iodide stained nucleiwere examined by flow cytometry. ModFit was used to generate the plots.The stained DNA profile is shown as the heavy line. The dark grey filledpeaks designate the proportions of the cell population at the G0/G1 (2n)and G2/M (4n) interphases of the cell cycle. The diagonal line filledpeak shows the S phase population. Quantitative values represent thepercent of the total cell population that is diploid G1, diploid G2 anddiploid S. The increase in the percent of the total cell populationaccumulated at the G2/M interphase, indicates that cell cycle arrestoccurred in all three types of epithelial cells following exposure tothe Cdt.

FIG. 16. Expression of the prominin-1 gene in epithelial cells. (FIG.16A) Real time (RT)-PGR of prominin-1 mRNA from the SCC cell linesRPMI2650, CAL-27 and FaDu and primary HGEC. PCR primers and conditionsare described in Materials and Methods. Values for the amount of PCRamplicon were normalized to that for the ubiquitous, constitutivelyexpressed TATA-binding protein (TBP) gene mRNA. Values were plottedrelative to that for Caco-2prominin-1 mRNA (positive control). The datawere plotted on two scales to depict the large difference between levelsofprominin-1 gene expression in the cell lines examined and arerepresented as mean values±SD; n=3 in each group. Asterisks markstatistically significant differences between the relative amount of theprominin-1 amplicon obtained from the particular SCC line mRNA and thatfrom HGEC mRNA (no expression). (FIG. 16A) Presence of CD133 in celllysates of SCC cells assessed by western blotting. CD133 was detectedwith an Enhanced Chemiluminescence Western Blotting Detection Kit(Amersham Pharmacia Biotech) after binding of CD 133/1 (AC133) mouseanti-human MAb (Miltenyi Biotec; 1:100) and horseradishperoxidase-conjugated anti-mouse IgG (Novagen; 1:3000). Prestainedmolecular weight standards were obtained from New England Biolabs.

FIG. 17. Heterogeneous distribution of CD133⁺ cells in SCC cellcultures. (FIG. 17A) CAL-27 and RPMI 2650 cells were stained with CD133/1 (AC133) mouse anti-human MAb (Miltenyi Biotec; 1:200) and goatanti-mouse IgG conjugated to Alexa Fluor (MAb-AF; Invitrogen/MolecularProbes® 1:400). Nuclei were stained with DAPI (blue fluorescence).Slides were viewed with a Nikon Eclipse 80i fluorescence microscope.Magnification 20× (CAL-27) and 40× (RPMI 2650). Insets 1 and 2 showenlarged images of stained cells. (FIG. 17B) RPMI 2650, FaDu, Caco-2 andprimary HGEC (1×10⁶ cells) examined by flow cytometry after stainingwith 100 ug of purified AC133.1 MAb IgG and Alexa Fluor 488 goatanti-mouse IgG (Invitrogen; 1:1000; red peaks) or Alexa Fluor 488conjugate alone (1:1000; green peaks). Control cells were not stained(blue peaks). RPMI 2650 cells were also stained with mouse anti-humanCD133/1 MAb conjugated to PE (Miltenyi Biotec; 1:100; inset; red peak).Gated runs were analyzed using FlowJo modeling software to obtain theplots. The number above each peak is the fluorescence value below which50% of the events were found (median). The pronounced increase influorescence intensity and relatively high median value indicate bindingof the AC133.1 MAb IgG and CD133.1 MAb-PE to RPMI 2650 and the positivecontrol (Caco-2).

FIG. 18. Construction and characterization of a mutated CdtA subunit fortargeting of prominin-1-expressing epithelial cells. (FIG. 18A) Thecrystal structure of the A. actinomycetemcomitans Cdt modeled with UCSFChimera and PDB file 2F2F. Essential active site residues H160 and H274in CdtB are shown in cyan. The four cysteine residues in the CdtAsubunit are displayed in magenta. The surface model of CdtA (inset) isrotated 90° counterclockwise, relative to that in the ribbon structure,to show both surface exposed C149 and C197 residues. Two predicteddisulfides C136/C149 and C178/C197 are marked by brackets and twosite-directed mutations resulted in the substitutions C149A and C178A.AC133.1 MAb IgG was attached to the surface-exposedC197 ofCdtA^(c149AC178A)-His₆ via SMPT. (FIG. 18B) Binding kinetics ofCdtA^(c149A) ^(̂) ^(C178A)-HiS6 determined in a CELISA. Increasingconcentrations of either wild-type CdtA-His6 or CdtA^(c149A, C178A)-His₆were added to immobilized RPMI 2650 and FaDu cells. Bound protein wasdetected with an anti-His>>Tag monoclonal antibody (Novagen; 1:3000),anti-mouse IgG horseradish peroxidase (Novagen; 1:3000) and horseradishperoxidase substrate ABTS-100 (Rdckland). Plates were read at awavelength of 405 nm in a Synergy 2 Microplate Reader (BjoTekInstruments, Inc.). Data are represented as mean values±SD; n=3 in eachgroup. Statistically significant differences were found between thebinding of the wild-type and mutated CdtA to RPMI 2650 (top asterisks)and FaDu (bottom asterisks). P<0.05. (C) Ability ofCdtA^(c149A, C178A)-His₆ (relative to wild-type CdtA) to form aheterotrimer with wild-type CdtB and CdtC determined by differentialdialysis. Aliquots of the reconstituted samples were examined on awestern blot before (BD) and after (AD) dialysis. The proteins weredetected by chemiluminescence after binding of anti-His>>Tag monoclonalantibody and horseradish peroxidase-conjugated anti-mouse IgG (Novagen;1:3000).

FIGS. 19A-D. Effect of CdtA^(c149A,c178A)BC-CD133MAb on the cell cycleof SCC cells. Caco-2, RPMI 2650 and FaDu cultures (90% confluent) weretreated with wild-type reconstituted recombinant Cdt (CdtABC), AC133.1MAb IgG-conjugated Cdt (CdtA^(c149A,C178A)BC-CD133MAb), AC133.1 MAb IgGalone (CD133MAb) or unconjugated mutated Cdt (CdtA^(c149A,C178A)BC) for36 h. Treated cultures received 10 μg/ml of protein/3.5×10⁶ cells. Noprotein was added to untreated control cultures. DNA profiles ofpropidium iodide stained nuclei were obtained by flow cytometry andModFit was used to construct the plots as described in the legend toFIG. 1. An increase in the percentage of the total cell populationaccumulated at the G2/M interphase was observed in RPMI 2650 culturestreated with wild-type Cdt and CdtA^(c149A,c178A)BC-CD133MAb. Similarresults were obtained with Caco-2. In the same experiment, RPMI 2650cultures were exposed to increasing concentrations ofCdtA^(c149A,c178A)BC-CD133MAb for 36 h and examined by flow cytometry.The percentage of cells in each treated culture having a 4n DNAconcentration (diploid G2) was plotted against the concentration of theCdt-MAb conjugate to obtain a dose response curve (last panel). The doseresponse was linear (dashed line) up to 16 ug/ml of protein/1×10⁶ cells(at time of toxin addition). The linear regression equation andR-squared value (square of the correlation coefficient) are shown.

FIG. 20. Stability of untreated gingival tissue in vitro. Explants wereincubated in culture medium at 37° C. in an atmosphere containing 10%CO₂ for (FIG. 20A) 0 h, (FIG. 20B) 18 h and (FIG. 20C) 24 h. Afterincubation the tissue was embedded and representative sections werestained with hematoxylin and eosin. Sections are shown at 4, 10 and 20×magnification. The red outline marks the enlarged area. GE, gingivalepithelium; RP, rete pegs; CT, connective tissue. Sections were alsoincubated with pan-keratin Ab3 mouse monoclonal antibody followed bygoat anti-mouse Alexa Fluor 488 to detect epithelial cells (greenfluorescence).

FIG. 21. Effects of Cdt on the integrity of gingival tissue. Explantswere treated with 10 μg/ml of CdtAB^(H160A)C for (FIG. 21A) 0 h, (FIG.21B) 18 h and (FIG. 21C) 36 h. Additional explants were treated withCdtABC: (FIG. 21D) 10 μg/ml for 0 h, (FIG. 21E) 10 μg/ml for 18 h and(F) 5 μg/ml for 36 h. Sections were stained with hematoxylin and eosinand depicted as described in the legend for FIG. 20. The insets areenlarged areas bounded by the small red outlines in the same images.

FIG. 22. Localization of CdtB in tissue treated with Cdt. Explants wereincubated in tissue culture medium for 19 h. (FIG. 22A) Tissue treatedwith 10 μg/ml of CdtABC, (FIG. 22B) Tissue treated with 10 μg/ml ofCdtAB^(H160A)C, (FIG. 22C) Untreated. The same sections were incubatedwith both pan-keratin Ab3 mouse antibody and rabbit anti-CdtB antibodyfollowed by goat anti-mouse Alexa Fluor 488 (green fluorescence) andgoat anti-rabbit IgG Alexa Fluor 594 conjugate (red fluorescence). Cellnuclei were visualized with DAPI (blue fluorescence). GS, gingivalsurface; GE, gingival epithelium; RP, rete pegs; CT, connective tissue.The arrows in (FIG. 22A) show CdtB in the various tissue layers. Theinset in (FIG. 22C) is a tissue section stained only with the goatanti-mouse Alexa Fluor 488 conjugate and DAPI.

FIG. 23. Effect of Cdt on cultured primary HGEC and HGF. (FIG. 23A) HGECincubated with mouse anti-human Ep-Cam (EBA-1) antibody and HGF treatedwith anti-fibroblast CD90/Thy-1 antigen (Ab-1) antibody. Bothpreparations were then stained with Alexa Fluor 488 goat anti-mouse IgGconjugate (green fluorescence). Nuclei were stained with DAPI. Thearrows show the presence of Ep-CAM on the cell surface. The inset showscells stained only with the goat anti-mouse Alexa Fluor 488 conjugate.(FIG. 23B) Flow cytometry of HGEC untreated and treated with 2.5 μg/mlof CdtABC for 36 h. The inset shows HGEC treated with 10 μg/ml ofCdtAB^(H160A)C for 36 h. HGF were untreated and treated with 10 μg/ml ofCdtABC for 96 h.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to recombinant polypeptides comprising a chimerathat inhibits cell proliferation and a method of using the same.

The invention provides, in one embodiment, a recombinant polypeptidecomprising a chimera, wherein said chimera comprises a DNase I fragmentand a Cdt fragment. In another embodiment, the chimera comprises a DNaseI fragment and a homologue of a Cdt fragment. In another embodiment, thechimera comprises a homologue of a DNase I fragment and a Cdt fragment.In another embodiment, the chimera comprises a homologue of a DNase Ifragment and a homologue of a Cdt fragment. In another embodiment, thepresent invention provides a recombinant polypeptide comprising achimera, wherein said chimera comprises a DNase I fragment or ahomologue thereof and a Cdt fragment or a homologue thereof. In anotherembodiment, said chimera comprises a Cdt subunit.

In one embodiment, DNase I is an endonuclease that nonspecificallycleaves DNA to release di-, tri- and oligonucleotide products with 5‘-phosphorylated and 3’-hydroxylated ends. In one embodiment, DNase Iacts on single- and double-stranded DNA, chromatin and RNA: DNA hybrids.In one embodiment, DNase I for use in the compositions and methods ofthe present invention is derived from humans. In one embodiment, DNase Ifor use in the compositions and methods of the present invention isderived from a murine species, which in one embodiment, is rat or mouse.In one embodiment, DNase I for use in the compositions and methods ofthe present invention is derived from Mus musculus, Homo sapiens, Bostaurus, Bacillus subtilis, Rattus norvegicus, Oryctolagus cuniculus,Salmo salar, Gallus gallus, Canis lupus familiaris, Rhodopirellulabaltica SH 1, Sus scrofa, Equus caballus, Dictyostelium discoideum,Pseudomonas putida GB-1, Pseudomonas putida W619, Azoarcus sp. BH72,Synechococcus sp., Bacteroides caccae ATCC 43185, Xenopus laevis, orAspergillus niger.

In one embodiment, a DNase I fragment of the present invention is anN-terminal fragment. In another embodiment, a DNase I fragment of thepresent invention is a C-terminal fragment.

In one embodiment, the nucleic acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 1) TTGTACAAAAAAGCAGGCTTGGAAGGAGTTCGAACCATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTATCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGTAGCTCGAGTGCGGCCGC AACCCAGCTTTCTTGTAC.In one embodiment, the coding sequence of the nucleic acid sequence isunderlined. In another embodiment, the DNase I fragment is a homologueof SEQ ID NO: 1. In another embodiment, the DNase I fragment is avariant of SEQ ID NO: 1. In another embodiment, the DNase I fragment isan isoform of SEQ ID NO: 1. In another embodiment, the DNase I fragmentis a fragment of SEQ ID NO: 1. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the nucleic acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 2) ATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTATCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGTAG.In another embodiment, the DNase I fragment is a homologue of SEQ ID NO:2. In another embodiment, the DNase I fragment is a variant of SEQ IDNO: 2. In another embodiment, the DNase I fragment is an isoform of SEQID NO: 2. In another embodiment, the DNase I fragment is a fragment ofSEQ ID NO: 2. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the nucleic acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 3) ATGGTGAGGGGAATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCTCGAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCTAAC AAAGCC.In another embodiment, the DNase I fragment is a homologue of SEQ ID NO:3. In another embodiment, the DNase I fragment is a variant of SEQ IDNO: 3. In another embodiment, the DNase I fragment is an isoform of SEQID NO: 3. In another embodiment, the DNase I fragment is a fragment ofSEQ ID NO: 3. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the amino acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 4) MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVSEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLK.In another embodiment, the DNase I fragment is a homologue of SEQ ID NO:4. In another embodiment, the DNase I fragment is a variant of SEQ IDNO: 4. In another embodiment, the DNase I fragment is an isoform of SEQID NO: 4. In another embodiment, the DNase I fragment is a fragment ofSEQ ID NO: 4. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the amino acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 5) MVRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKLEHHHHHHHMLEDPAAN KA.In another embodiment, the DNase I fragment is a homologue of SEQ ID NO:5. In another embodiment, the DNase I fragment is a variant of SEQ IDNO: 5. In another embodiment, the DNase I fragment is an isoform of SEQID NO: 5. In another embodiment, the DNase I fragment is a fragment ofSEQ ID NO: 5. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the amino acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 6) MVRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQIMSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKLEHHHHHHHMLEDPAAN KA.In another embodiment, the DNase I fragment is a homologue of SEQ ID NO:6. In another embodiment, the DNase I fragment is a variant of SEQ IDNO: 6. In another embodiment, the DNase I fragment is an isoform of SEQID NO: 6. In another embodiment, the DNase I fragment is a fragment ofSEQ ID NO: 6. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the amino acid sequence encoding the DNase Ifragment is:

(SEQ ID NO: 7) MVRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQIMSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKLEHHHHHHHMLEDPAAN KA.In another embodiment, the DNase I fragment is a homologue of SEQ ID NO:7. In another embodiment, the DNase I fragment is a variant of SEQ IDNO: 7. In another embodiment, the DNase I fragment is an isoform of SEQID NO: 7. In another embodiment, the DNase I fragment is a fragment ofSEQ ID NO: 7. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a DNase I, or its fragment or homologue comprisesa mutated form of a DNase I. In another embodiment, a DNase I, or itsfragment or homologue comprises a mutated form of a DNase I fragment. Inanother embodiment, a mutated DNase I, a mutated DNase I fragmentcomprises a deletion mutation. In another embodiment, a mutated DNase I,a mutated CdtB toxin fragment comprises an insertion mutation. Inanother embodiment, a mutated DNase I, a mutated DNase I fragmentcomprises a substitution mutation.

In one embodiment, a chimera of the present invention comprises a DNasefragment. In another embodiment, a chimera of the present inventioncomprises a nuclease fragment. In another embodiment, a chimera of thepresent invention comprises an RNase fragment.

In one embodiment, a DNase for use in the compositions and methods ofthe present invention is an exodeoxyribonuclease, and, in oneembodiment, cleaves only residues at the ends of DNA molecules. Inanother embodiment, a DNase for use in the compositions and methods ofthe present invention is an endodeoxyribonuclease, and, in oneembodiment, cleaves anywhere along the polynucleotide chain. In oneembodiment, a DNase for use in the compositions and methods of thepresent invention is deoxyribonuclease I or, in another embodiment,deoxyribonuclease II.

In another embodiment, the chimera provided herein is a Cdt-DNase Ichimera. In another embodiment, the chimera provided herein is amultimeric chimera protein comprising DNase I or its fragment orhomologue; and a Cdt toxin, or its fragment or homologue. In anotherembodiment, the chimera provided herein comprises DNase I or itsfragment or homologue; and a Cdt toxin. In another embodiment, thechimera provided herein is a multimeric chimera protein comprising DNaseI and a Cdt toxin, or its fragment or homologue. In another embodiment,the chimera provided herein consists DNase I or its fragment orhomologue; and a Cdt toxin.

In one embodiment, a Cdt fragment of the present invention is anN-terminal fragment. In another embodiment, a Cdt fragment of thepresent invention is a C-terminal fragment.

In one embodiment, a CdtA, CdtB, CdtC, Cdt holotoxin, or fragmentthereof used in the compositions and methods of the present invention isderived from Escherichia coli, Campylobacter jejuni, Campylobacterupsaliensis, Campylobacter coli, Shigella dysenteriae, Haemophilusducreyi, Helicobacter hepaticus, Helicobacter flexispira, Helicobacterbilis, Helicobacter canis, Aggregatibacter actinomycetemcomitans, orActinobacillus actinomycetemcomitans.

In one embodiment, the Cdts are a family of heat-labile proteincytotoxins produced by several different bacterial species includingdiarrheal disease-causing enteropathogens such as some Escherichia coliisolates, Campylobacter jejuni, Shigella species, Haemophilus ducreyiand Actinobacillus actinomycetemcomitans. Thus, in one embodiment, a Cdtsubunit of the present invention is derived from Actinobacillusactinomycetemcomitans. In another embodiment, a Cdt subunit of thepresent invention is derived from Escherichia coli isolates,Campylobacter jejuni, Shigella species, or Haemophilus ducreyi.

Cdts are encoded by three genes, designated cdtA, cdtB, and cdtC whichare arranged as an apparent operon. These three genes specify threepolypeptides designated CdtA, CdtB and CdtC with apparent molecularmasses of 28, 32 and 20 kDa, respectively, that form a heterotrimericholotoxin. Several cell lines and cell types have been shown to besensitive to Cdt including human lymphoid cells, fibroblasts, humanembryonic intestinal epithelial cells, a human colon carcinoma cellline, and human keratinocytes, among others. In one embodiment,lymphocytes are the most sensitive (4-5 fold) to Cdt, suggesting thatCdt most likely functions as an immunotoxin. In response to Cdt,proliferating cells exhibit G2 arrest and eventually cell deathresulting from activation of the apoptotic cascade. In anotherembodiment, the cell cycle arrest results in a cessation of celldivision. In one embodiment, the Cdts produce other effects, including,in another embodiment, progressive cellular distention.

The heterotrimeric Cdt holotoxin functions as an AB2 toxin where CdtB isthe active (A) unit, and the complex of CdtA and CdtC comprise thebinding (B) unit. In one embodiment, the CdtA and CdtC subunits areinvolved in the adhesion to target cells. In one embodiment, CdtA andCdtC are required for the toxin to associate with lipid microdomainswithin lymphocyte membranes. In one embodiment, the CdtC subunitinteracts specifically with cholesterol. In one embodiment, the CdtBmust be internalized and associate with cellugyrin, a microvessicleassociated protein, in order to induce cell cycle arrest. In oneembodiment, CdtB acts as a PIP3 phosphatase depleting cells of PIP3 andthereby blocking the Akt survival/proliferation pathway. In oneembodiment, CdtB does not act on phosphatides or proteins. In oneembodiment, the Cdt holotoxin has structural homology with inositolpolyphosphate 5-phosphatase. In another embodiment, the CdtB subunit hastype I deoxyribonuclease-like activity. In another embodiment, the Bsubunit has endonuclease activity that results in double strand breaksand blunt ends. In one embodiment, the Cdt holotoxin has structuralhomology with DNAse I.

In another embodiment, CdtA is encoded by the following nucleic acidsequence:

(SEQ ID NO: 8) ATGGCTCCGAGGAGAGGTACAATGAAAAAGTTTTTACCTGGTCTTTTATTGATGGGTTTAGTGGCTTGTTCGTCAAATCAACGAATGAGTGACTATTCTCAGCCTGAATCTCAATCTGATTTAGCACCTAAATCTTCAACAACACAATTCCAACCCCAACCCCTATTATCAAAAGCATCTTCAATGCCATTGAATTTGCTCTCTTCATCCAAGAATGGACAGGTATCGCCGTCTGAACCATCAAACTTTATGACTTTGATGGGACAAAATGGGGCACTGTTGACTGTCTGGGCGCTAGCAAAACGCAATTGGTTATGGGCTTATCCCAATATATATTCGCAGGACTTTGGAAATATTCGTAATTGGAAGATAGAACCTGGTAAACACCGTGAATATTTTCGTTTTGTTAATCAATCTTTAGGTACATGTATTGAAGCTTACGGTAATGGTTTAATTCATGATACTTGTAGTCTGGACAAATTAGCACAAGAGTTTGAGTTATTACCTACTGATAGTGGTGCGGTTGTCATTAAAAGTGTGTCACAAGGACGTTGTGTCACTTATAATCCTGTAAGTCCAACATATTATTCAACAGTTACATTATCAACTTGTGATGGCGCAACAGAACCATTACGTGATCAAACATGGTATCTCGCTCCTCCTGTATTAGAAGCAACAGCGGTTAATCACCACCACCACCACCACGGATCCGGGCTGCTAACAAAGCCCCGAAAGGAAGC.In another embodiment, the CdtA subunit is a homologue of SEQ ID NO: 8.In another embodiment, the CdtA subunit is a variant of SEQ ID NO: 8. Inanother embodiment, the CdtA subunit is an isoform of SEQ ID NO: 8. Inanother embodiment, the CdtA subunit is a fragment of SEQ ID NO: 8. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, CdtA is encoded by the following amino acidsequence:

(SEQ ID NO: 9) MKKFLPGLLLMGLVACSSNQRMSDYSQPESQSDLAPKSSTTQFQPQPLLSKASSMPLNLLSSSKNGQVSPSEPSNFMTLMGQNGALLTVWALAKRNWLWAYPNIYSQDFGNIRNWKIEPGKHREYFRFVNQSLGTCIEAYGNGLIHDTCSLDKLAQEFELLPTDSGAVVIKSVSQGRCVTYNPVSPTYYSTVTLSTCDGATEPLRDQTWYLAPPVLEATAVNHHHHHHGSGLLTKPRKE.In another embodiment, the CdtA subunit is a homologue of SEQ ID NO: 9.In another embodiment, the CdtA subunit is a variant of SEQ ID NO: 9. Inanother embodiment, the CdtA subunit is an isoform of SEQ ID NO: 9. Inanother embodiment, the CdtA subunit is a fragment of SEQ ID NO: 9. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the amino acid sequence of a CdtA subunit of thepresent invention is:

(SEQ ID NO: 10) LLSSSKNGQVSPSEPSNFMTLMGQNGALLTVWALAKRNWLWAYPNIYSQDFGNIRNWKIEPGKHREYFRFVNQSLGTCIEAYGNGLIHDTCSLDKLAQEFELLPTDSGAVVIKSVSQGRCVTYNPVSPTYYSTVTLSTCDGATEPLRDQT WYLAPPVLEATAV.In another embodiment, the CdtA subunit is a homologue of SEQ ID NO: 10.In another embodiment, the CdtA subunit is a variant of SEQ ID NO: 10.In another embodiment, the CdtA subunit is an isoform of SEQ ID NO: 10.In another embodiment, the CdtA subunit is a fragment of SEQ ID NO: 10.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the CdtA subunit has an amino acid sequence setforth in one of the following GenBank entries: AAF81760; AAF19157;AAD10621; AAB06707; AAA18785; NP_(—)860977; AAP78043; YP_(—)002343541;CAL34252; Q46668; AAT92047; AAC70897; AAZ16246; ABV51672.1;YP_(—)001272540.1; BAF63360.1; YP_(—)999805.1; ZP_(—)02270538.1;AAB06707.1; EAQ71960.1; ABJ00842.1; YP_(—)178099.1; AAW34670.1;NP_(—)873397.1; or AAP95786.1. In another embodiment, the CdtA subunithas any CdtA subunit amino acid sequence known in the art. In anotherembodiment, the CdtA subunit is a homologue of a sequence from one ofthe above GenBank entries. In another embodiment, the CdtA subunit is avariant of a sequence from one of the above GenBank entries. In anotherembodiment, the CdtA subunit is an isoform of a sequence from one of theabove GenBank entries. In another embodiment, the CdtA subunit is afragment of a sequence from one of the above GenBank entries. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the nucleotide sequence of a CdtA subunit of thepresent invention is:

(SEQ ID NO: 11) ttgctctcttcatccaagaatggacaggtatcgccgtctgaaccatcaaactttatgactttgatgggacaaaatggggcactgttgactgtctgggcgctagcaaaacgcaattggttatgggcttatcccaatatatattcgcaggactttggaaatattcgtaattggaagatagaacctggtaaacaccgtgaatattttcgttttgttaatcaatctttaggtacatgtattgaagcttacggtaatggtttaattcatgatacttgtagtctggacaaattagcacaagagtttgagttattacctactgatagtggtgcggttgtcattaaaagtgtgtcacaaggacgttgtgtcacttataatcctgtaagtccaacatattattcaacagttacattatcaacttgtgatggcgcaacagaaccattacgtgatcaaacatggtatctcgctcctcctgtattagaagcaacagcggtt.In another embodiment, the nucleotide sequence of the CdtA subunit is ahomologue of SEQ ID NO: 11. In another embodiment, the nucleotidesequence of the CdtA subunit is a variant of SEQ ID NO: 11. In anotherembodiment, the nucleotide sequence of the CdtA subunit is an isoform ofSEQ ID NO: 11. In another embodiment, the nucleotide sequence of theCdtA subunit is a fragment of SEQ ID NO: 11. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the CdtA subunit has a nucleic acid sequence setforth in one of the following GenBank entries: AL111168.1; AE017125.1;CP000814.1; AB285204.1; NZ_AASL01000001.1; U51121.1; CP000538.1;CP000468.1; CP000025.1; or AE017143.1. In another embodiment, the CdtAsubunit has any CdtA subunit nucleic acid sequence known in the art. Inanother embodiment, the CdtA subunit is a homologue of a sequence fromone of the above GenBank entries. In another embodiment, the CdtAsubunit is a variant of a sequence from one of the above GenBankentries. In another embodiment, the CdtA subunit is an isoform of asequence from one of the above GenBank entries. In another embodiment,the CdtA subunit is a fragment of a sequence from one of the aboveGenBank entries. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, CdtA binds to a specific cell receptor, while inanother embodiment, CdtA stabilizes the holotoxin. In one embodiment,analysis of the crystal structure of the toxin suggest that CdtAcontains ricin-like domains, leading investigators to propose that itassociates with carbohydrate.

In a particular embodiment, the invention provides a recombinant CdtApolypeptide comprising at least one of the mutations C149A and C178A. Inanother embodiment, the invention provides a recombinant CdtApolypeptide comprising the mutations C149A and C178A. In yet anotherembodiment, the invention provides a nucleic acid sequence encoding (i)a CdtA polypeptide comprising at least one of the mutations C149A andC178A or (ii) a nucleic acid sequence that is at least 85% identical tothe sequence of (i).

In another embodiment, the invention provides at least one of themutations C149A and C178A in SEQ ID NO: 9. In another embodiment, theinvention provides the mutations C149A and C178A in SEQ ID NO: 9. In yetanother embodiment, the invention provides a nucleic acid sequenceencoding (i) a CdtA polypeptide comprising at least one of the mutationsC149A and C178A (e.g., mutations in SEQ ID NO: 9) or (ii) a nucleic acidsequence that is at least 85% identical to the sequence of (i).

In another embodiment, the invention provides a composition comprising:a recombinant CdtA polypeptide comprising at least one of the mutationsC149A and C178A, said polypeptide operably linked to a ligand that bindsspecifically to an antigen expressed on the surface of a cancerousepithelial cell type.

In another embodiment, the invention provides a recombinant CdtApolypeptide operably linked to a ligand that binds specifically to anantigen expressed on the surface of a cancerous epithelial cell type,wherein said recombinant CdtA polypeptide comprises at least one of themutations C149A and C178A.

In another embodiment, the invention provides a chimeric CdtApolypeptide comprising at least one of the mutations C149A and C178A.

In another embodiment, the invention provides an isolated nucleic acidsequence encoding (i) a CdtA polypeptide comprising at least one of themutations C149A and C178A or (ii) a nucleic acid sequence that is atleast 85% identical to the sequence of (i).

In one embodiment, the amino acid sequence of a CdtB subunit of thepresent invention is:

(SEQ ID NO: 12) NLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADILMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRSRPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHALATGGSDAVSLIRNIFTTFTSSPSSPERRGYSWMVVGDFNRAPVNLEAALRQEPAVSENTMAPTEPTHRSGNILDYAILHDAHLPRREQARERIGASLMLNQLRSQITSDH FPVSFVRDR.In another embodiment, the CdtB subunit is a homologue of SEQ ID NO: 12.In another embodiment, the CdtB subunit is a variant of SEQ ID NO: 12.In another embodiment, the CdtB subunit is an isoform of SEQ ID NO: 12.In another embodiment, the CdtB subunit is a fragment of SEQ ID NO: 12.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the CdtB subunit has an amino acid sequence setforth in one of the following GenBank entries: ZP_(—)01072217;NP_(—)860978; YP_(—)002343540; YP_(—)002308521; ZP_(—)03223221;NP_(—)873398; YP_(—)001481648; YP_(—)852557; YP_(—)999804; YP_(—)434821;YP_(—)178098; YP_(—)001272541; ZP_(—)01100899; ZP_(—)01067880;ZP_(—)00370497; or ZP_(—)00369375. In another embodiment, the CdtBsubunit has any CdtB subunit amino acid sequence known in the art. Inanother embodiment, the CdtB subunit is a homologue of a sequence fromone of the above GenBank entries. In another embodiment, the CdtBsubunit is a variant of a sequence from one of the above GenBankentries. In another embodiment, the CdtB subunit is an isoform of asequence from one of the above GenBank entries. In another embodiment,the CdtB subunit is a fragment of a sequence from one of the aboveGenBank entries. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, the nucleotide sequence of a CdtB subunit of thepresent invention is:

(SEQ ID NO: 13) aacttgagtgatttcaaagtagcaacttggaatctgcaaggttcttcagctgtaaatgaaagtaaatggaatattaatgtgcgccaattattatcgggagaacaaggtgcagatattttgatggtacaagaagcgggttcattaccaagttcggcagtaagaacctcacgagtaattcaacatgggggaacgccaattgaggaatatacctggaatttaggtactcgctcccgtccaaatatggtctatatttattattcccgtttagatgttggggcaaaccgagtgaacttagctatcgtgtcacgtcgtcaagccgatgaagcttttatcgtacattctgattcttctgtgcttcaatctcgcccggcagtaggtatccgcattggtactgatgtattttttacagtgcatgctttggccacaggtggttctgatgcggtaagtttaattcgtaatatcttcactacttttacctcatcaccatcatcaccggaaagacgaggatatagctggatggttgttggtgatttcaatcgtgcgccggttaatctggaagctgcattaagacaggaacccgccgtgagtgaaaatacaattattattgcgccaacagaaccgactcatcggtccggtaatattttagattatgcgattttacatgacgcacatttaccacgtcgagagcaagcacgtgaacgtatcggcgcaagtttaatgttaaatcagttacgctcacaaattacatccgatcattttcctgttagttttgttcgtgatc.In another embodiment, the nucleotide sequence of the CdtB subunit is ahomologue of SEQ ID NO: 13. In another embodiment, the nucleotidesequence of the CdtB subunit is a variant of SEQ ID NO: 13. In anotherembodiment, the nucleotide sequence of the CdtB subunit is an isoform ofSEQ ID NO: 13. In another embodiment, the nucleotide sequence of theCdtB subunit is a fragment of SEQ ID NO: 13. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the CdtB subunit has a nucleic acid sequence setforth in one of the following GenBank entries: AL111168.1; AL627271.1;AE017125.1; CP000814.1; AB285204.1; EU794049.1; DQ092613.1; CP000468.1;CP000026.1; CP000155.1; CP000025.1; AE017143.1; CP000538.1; U51121.1;NZ_AASL01000001.1; or AE014613.1. In another embodiment, the CdtBsubunit has any CdtB subunit nucleic acid sequence known in the art. Inanother embodiment, the CdtB subunit is a homologue of a sequence fromone of the above GenBank entries. In another embodiment, the CdtBsubunit is a variant of a sequence from one of the above GenBankentries. In another embodiment, the CdtB subunit is an isoform of asequence from one of the above GenBank entries. In another embodiment,the CdtB subunit is a fragment of a sequence from one of the aboveGenBank entries. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, compositions and methods of the present inventioncomprise or use a Cdt fragment. In one embodiment, the Cdt fragment is aCdtB fragment or a homologue thereof.

In another embodiment, the amino acid sequence encoding the CdtBfragment is:

(SEQ ID NO: 14) MQWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADILMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRSRPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHALATGGSDAVSLIRNIFTTFTSSPSSPERRGYSWMVVGDFNRAPVNLEAALRQEPAVSENTIIIAPTEPTHQSGNILDYAILHDAHLPRREQVRERIGASLMLNQLRSQITSDHFP.In another embodiment, the CdtB fragment is a homologue of SEQ ID NO:14. In another embodiment, the CdtB fragment is a variant of SEQ ID NO:14. In another embodiment, the CdtB fragment is an isoform of SEQ ID NO:14. In another embodiment, the CdtB fragment is a fragment of SEQ ID NO:14. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the nucleic acid sequence encoding the CdtBfragment is:

(SEQ ID NO: 15) ATGCAATGGGTAAAGCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGGGAGAACAAGGTGCAGATATTTTGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTCCCGTCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCGTACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCTTTGGCCACAGGTGGTTCTGATGCGGTAAGTTTAATTCGTAATATCTTCACTACTTTTACCTCATCACCATCATCACCGGAAAGACGAGGATATAGCTGGATGGTTGTTGGTGATTTCAATCGTGCGCCGGTTAATCTGGAAGCTGCATTAAGACAGGAACCCGCCGTGAGTGAAAATACAATTATTATTGCGCCAACAGAACCGACTCATCAGTCCGGTAATATTTTAGATTATGCGATTTTACATGACGCACATTTACCACGTCGAGAGCAAGTACGTGAACGTATCGGCGCAAGTTTAATGTTAAATCAGTTACGCTCACAAATTACATCCGATCATTTTCCT.In another embodiment, the CdtB fragment is encoded by a homologue ofSEQ ID NO: 15. In another embodiment, the CdtB fragment is encoded by avariant of SEQ ID NO: 15. In another embodiment, the CdtB fragment isencoded by an isoform of SEQ ID NO: 15. In another embodiment, the CdtBfragment is encoded by a fragment of SEQ ID NO: 15. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the amino acid sequence encoding the CdtBfragment is:

(SEQ ID NO: 16) MQWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADILMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRSRPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHALATGGSDAVSLIRNIFTTFTSSPSSPERRGYSWMVVGDFNRAPVNLEAALRQEPAVSENTIIIAPTEPTHQSGNILDYAILHDAHLPRREQVRERIGASLMLNQLRSQITSDHFPVSFVHDRHHHHHHGSGC.In another embodiment, the CdtB fragment is a homologue of SEQ ID NO:16. In another embodiment, the CdtB fragment is a variant of SEQ ID NO:16. In another embodiment, the CdtB fragment is an isoform of SEQ ID NO:16. In another embodiment, the CdtB fragment is a fragment of SEQ ID NO:16. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a CdtB toxin, or its fragment or homologuecomprises a mutated form of a CdtB toxin. In another embodiment, a CdtBtoxin, or its fragment or homologue comprises a mutated form of a CdtBtoxin fragment. In another embodiment, a mutated CdtB toxin or a mutatedCdtB toxin fragment comprises a deletion mutation. In anotherembodiment, a mutated CdtB toxin or a mutated CdtB toxin fragmentcomprises an insertion mutation. In another embodiment, a mutated CdtBtoxin or a mutated CdtB toxin fragment comprises a substitutionmutation.

In one embodiment, a CdtB fragment of the present invention comprises amutation. In one embodiment, the mutation adds a DNA contact residue. Inone embodiment, the mutation is G55R, S99Y, V134R, or a combinationthereof. In another embodiment, the mutation adds a metal ion bindingresidue. In one embodiment, the mutation is L62E. In another embodiment,the mutation adds a residue that hydrogen bonds to a catalytichistidine, which in one embodiment, is H160. In one embodiment, themutation is R100E. In another embodiment, the mutation is Y174P. In oneembodiment, the mutation is a combination of two or more of themutations described hereinabove.

In another embodiment, cdtC is encoded by the following nucleic acidsequence:

(SEQ ID NO: 17) ATGGTCGCTAAGGAGAATACTATGAAAAAATATTTATTGAGCTTCTTATTAAGCATGATATTGACTTTGACGAGTCATGCAGAATCAAATCCTGATCCGACTACTTATCCTGATGTAGAGTTATCGCCTCCTCCACGTATTAGCTTGCGTAGTTTGCTTACGGCTCAACCAATTAAAAATGACCATTATGATTCACATAATTATTTAAGTACACATTGGGAATTAATTGATTACAAGGGAAAAGAATATGAAAAATTACGTGACGGTGGTACGTTGGTTCAATTTAAAGTGGTCGGTGCAGCAAAATGTTTTGCTTTCCCAGGCGAAGGCACAACTGATTGTAAAGATATTGATCATACTGTGTTTAACCTTATTCCAACTAATACAGGTGCGTTTTTAATCAAAGATGCCCTATTAGGATTTTGTATGACAAGCCATGACTTTGATGATTTGAGGCTTGAACCTTGTGGAATTTCAGTGAGTGGTCGAACCTTTTCGTTGGCGTATCAATGGGGAATATTACCTCCTTTTGGGCCAAGTAAAATTTTAAGACCACCGGTGGGGAGAAATCAGGGTAGCCACCACCACCACCACCACGGA TCCGGCTGCTAA.In another embodiment, the nucleic acid encoding the CdtC subunit is ahomologue of SEQ ID NO: 17. In another embodiment, the nucleic acidencoding the CdtC subunit is a variant of SEQ ID NO: 17. In anotherembodiment, the nucleic acid encoding the CdtC subunit is an isoform ofSEQ ID NO: 17. In another embodiment, the nucleic acid encoding the CdtCsubunit is a fragment of SEQ ID NO: 17. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, CdtC is encoded by the following amino acidsequence:

(SEQ ID NO: 18) MVAKENTMKKYLLSFLLSMILTLTSHAESNPDPTTYPDVELSPPPRISLRSLLTAQPIKNDHYDSHNYLSTHWELIDYKGKEYEKLRDGGTLVQFKVVGAAKCFAFPGEGTTDCKDIDHTVFNLIPTNTGAFLIKDALLGFCMTSHDFDDLRLEPCGISVSGRTFSLAYQWGILPPFGPSKILRPPVGRNQGSHHHHHHG SGC.In another embodiment, the CdtC subunit is a homologue of SEQ ID NO: 18.In another embodiment, the CdtC subunit is a variant of SEQ ID NO: 18.In another embodiment, the CdtC subunit is an isoform of SEQ ID NO: 18.In another embodiment, the CdtC subunit is a fragment of SEQ ID NO: 18.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, CdtC is encoded by the following amino acidsequence:

(SEQ ID NO: 19) MKKYLLSFLLSMILTLTSHAESNPDPTTYPDVELSPPPRISLRSLLTAQPIKNDHYDSHNYLSTHWELIDYKGKEYEKLRDGGTLVQFKVVGAAKCFAFPGEGTTDCKDIDHTVFNLIPTNTGAFLIKDALLGFCMTSHDFDDLRLEPCGISVSGRTFSLAYQWGILPPFGPSKILRPPVGRNQGSHHHHHHGSGC.In another embodiment, the CdtC subunit is a homologue of SEQ ID NO: 19.In another embodiment, the CdtC subunit is a variant of SEQ ID NO: 19.In another embodiment, the CdtC subunit is an isoform of SEQ ID NO: 19.In another embodiment, the CdtC subunit is a fragment of SEQ ID NO: 19.Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, the amino acid sequence of a CdtC subunit of thepresent invention is:

(SEQ ID NO: 20) ESNPDPTTYPDVELSPPPRISLRSLLTAQPIKNDHYDSHNYLSTHWELIDYKGKEYEKLRDGGTLVQFKVVGAAKCFAFPGEGTTDCKDIDHTVFNLIPTNTGAFLIKDALLGFCMTSHDFDDLRLEPCGISVSGRTFSLAYQWGILPPF GPSKILRPPVGRNQGS.In another embodiment, the CdtC subunit is a homologue of SEQ ID NO: 20.In another embodiment, the CdtC subunit is a variant of SEQ ID NO: 20.In another embodiment, the CdtC subunit is an isoform of SEQ ID NO: 20.In another embodiment, the CdtC subunit is a fragment of SEQ ID NO: 20.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the CdtC subunit has an amino acid sequence setforth in one of the following GenBank entries: YP_(—)002343539.1;NP_(—)860979.1; YP_(—)001481647.1; YP_(—)001272542.1; YP_(—)852558.1;YP_(—)999803.1; YP_(—)178097.1; NP_(—)873399.1; CAL34250.1; AAP78045.1;ABV51670.1; BAF63362.1; ABJ00844.1; EAQ72030.1; AAB06709.1;ZP_(—)02270536.1; or AAW34668.1. In another embodiment, the CdtC subunithas any CdtC subunit amino acid sequence known in the art. In anotherembodiment, the CdtC subunit is a homologue of a sequence from one ofthe above GenBank entries. In another embodiment, the CdtC subunit is avariant of a sequence from one of the above GenBank entries. In anotherembodiment, the CdtC subunit is an isoform of a sequence from one of theabove GenBank entries. In another embodiment, the CdtC subunit is afragment of a sequence from one of the above GenBank entries. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the nucleotide sequence of a CdtC subunit of thepresent invention is:

(SEQ ID NO: 21) gaatcaaatcctgatccgactacttatcctgatgtagagttatcgcctcctccacgtattagcttgcgtagtttgcttacggctcaaccaattaaaaatgaccattatgattcacataattatttaagtacacattgggaattaattgattacaagggaaaagaatatgaaaaattacgtgacggtggtacgttggttcaatttaaagtggtcggtgcagcaaaatgttttgctttcccaggcgaaggcacaactgattgtaaagatattgatcatactgtgtttaaccttattccaactaatacaggtgcgtttttaatcaaagatgccctattaggattttgtatgacaagccatgactttgatgatttgaggcttgaaccttgtggaatttcagtgagtggtcgaaccttttcgttggcgtatcaatggggaatattacctccttttgggccaagtaaaattttaagaccaccggtggggagaaatcagggtagc.In another embodiment, the nucleotide sequence of the CdtC subunit is ahomologue of SEQ ID NO: 21. In another embodiment, the nucleotidesequence of the CdtC subunit is a variant of SEQ ID NO: 21. In anotherembodiment, the nucleotide sequence of the CdtC subunit is an isoform ofSEQ ID NO: 21. In another embodiment, the nucleotide sequence of theCdtC subunit is a fragment of SEQ ID NO: 21. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the CdtC subunit has a nucleic acid sequence setforth in one of the following GenBank entries: AL111168.1; AE017125.1;CP000814.1; AB285204.1; CP000468.1; CP000538.1; U51121.1;NZ_AASL01000001.1; or CP000025.1. In another embodiment, the CdtCsubunit has any CdtC subunit nucleic acid sequence known in the art. Inanother embodiment, the CdtC subunit is a homologue of a sequence fromone of the above GenBank entries. In another embodiment, the CdtCsubunit is a variant of a sequence from one of the above GenBankentries. In another embodiment, the CdtC subunit is an isoform of asequence from one of the above GenBank entries. In another embodiment,the CdtC subunit is a fragment of a sequence from one of the aboveGenBank entries. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, CdtC comprises a cholesterol recognition site and, inone embodiment, CdtC binds to both cell and model membranes in acholesterol dependent manner.

In another embodiment, the chimera provided herein is a CdtB-DNase Ichimera. In another embodiment, the chimera provided herein is amultimeric chimera protein comprising DNase I or its fragment orhomologue; and a CdtB toxin, or its fragment or homologue. In anotherembodiment, the chimera provided herein comprises DNase I or itsfragment or homologue; and a CdtB toxin. In another embodiment, thechimera provided herein is a multimeric chimera protein comprising DNaseI and a CdtB toxin, or its fragment or homologue. In another embodiment,the chimera provided herein consists of DNase I or its fragment orhomologue; and a CdtB toxin.

In one embodiment, a chimera of the present invention comprises (a) saidCdtB fragment in the amino terminus of said chimera and said DNase Ifragment in the carboxy terminus of said chimera or (b) a homologue of aDNase I fragment in the carboxy terminus of said chimera and a Cdtfragment in the amino terminus of said chimera or (c) a DNase I in thecarboxy terminus of said chimera and a homologue of a Cdt fragment inthe amino terminus of said chimera or (d) a homologue of a DNase Ifragment in the carboxy terminus of said chimera and a homologue of aCdt fragment in the amino terminus of said chimera.

In another embodiment, the DNase I is a clone of the DNase I gene fromHomo sapiens chromosome 16 (GenBank accession number AC005203). Inanother embodiment, the cdtB gene is from the human periodontalbacterium Actinobacillus actinomycetemcomitans Y4 (GenBank accessionnumber AF006830), which in another embodiment is called Aggregatibacteractinomycetemcomitans. In another embodiment, a unique SphI restrictionendonuclease cleavage site at about the midpoint of each sequence isused to construct two chimeric genes each containing opposite halves ofthe cdtB and DNase I genes.

In another embodiment, the nucleic acid sequence encoding the chimerais:

(SEQ ID NO: 22) TGCTAAATTCCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGTGAGGGGAATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCATGCTTTGGCCACAGGTGGTTCTGATGCGGTAAGTTTAATTCGTAATATCTTCACTACTTTTACCTCGTCACCATCATCACCGGAAAGACGAGGATATAGCTGGATGGTTGTTGGTGATTTCAATCGTGCGCCGGTTAATCTGGAAGCTGCATTAAGACAGGAACCCGCCGTGAGTGAAAATACAATTATTATTGCGCCAACAGAACCGACTCATCAGTCCGGTAATATTTTAGATTATGCGATTTTACATGACGCACATTTACCACGTCGAGAGCAAGTACGTGAACGTATCGGCGCAAGTTTAATGTTAAATCAGTTACGCTCACAAATTACATCCGATCATTTTCCTGTTAGTTTTGTTCATGATCGCCACCACCACCACCACCACGGATCCGGCTGCTAA.In one embodiment, the coding sequence of the nucleic acid sequence isunderlined. In another embodiment, the nucleic acid encoding the chimerais a homologue of SEQ ID NO: 22. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 22. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 22. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 22. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the nucleic acid sequence encoding the chimerais:

(SEQ ID NO: 23) ATGCAATGGGTAAAGCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGGGAGAACAAGGTGCAGATATTTTGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTCCCGTCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCGTACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGGGATCCCGGGAGCTCGTGGATCCGAATTCTGTACAGGCGCGCCTGCAGGACGTCGACGGTACCATCGATACGCGTTCGAAGCTTGCGGCCGCACAGCTGTATACACGTGCAAGCCAGCCAGAACTCGCTCCTGAAGACCCAGAGGATCTCGAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCTAACAAAGCCCGAAAAGAAGG.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 23. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 23. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 23. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 23. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the amino acid sequence encoding the chimera is:

(SEQ ID NO: 24) MVRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHALATGGSDAVSLIRNIFTTFTSSPSSPERRGYSWMVVGDFNRAPVNLEAALRQEPAVSENTIIIAPTEPTHQSGNILDYAILHDAHLPRREQVRERIGASLMLNQLRSQITSDHFPVSFVHDRHHHHHHGSGC.In another embodiment, the chimera is a homologue of SEQ ID NO: 24. Inanother embodiment, the chimera is a variant of SEQ ID NO: 24. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 24. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 24. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the amino acid sequence encoding the chimera is:

(SEQ ID NO: 25) MQWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADILMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRSRPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKGSRELVDPNSVQARLQDVDGTIDTRSKLAAAQLYTRASQPELAPEDPEDLEHHHHHHHMLEDPA ANKARKEG.In another embodiment, the chimera is a homologue of SEQ ID NO: 25. Inanother embodiment, the chimera is a variant of SEQ ID NO: 25. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 25. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 25. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the amino acid sequence encoding the chimera is:

(SEQ ID NO: 26) MQWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADILMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRSRPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKHHHHHHHMLEDPAA NKAQKK.In another embodiment, the chimera is a homologue of SEQ ID NO: 26. Inanother embodiment, the chimera is a variant of SEQ ID NO: 26. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 26. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 26. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the amino acid sequence encoding the chimera is:

(SEQ ID NO: 27) MVRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHALATGGSDAVSLIRNIFTTFTSSPSSPERRGYSWMVVGDFNRAPVNLEAALRQEPAVSENTIIIAPTEPTHQSGNILDYAILHDAHLPRREQVRERIGASLMLNQLRSQITSDHFPVSFVHDRHHHHHHGSGC.In another embodiment, the chimera is a homologue of SEQ ID NO: 27. Inanother embodiment, the chimera is a variant of SEQ ID NO: 27. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 27. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 27. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment a chimera of the present invention binds to both CdtAand CdtC. In another embodiment, a chimera of the present inventionbinds to CdtC only.

In another embodiment, the chimera is encoded by the following nucleicacid sequence:

(SEQ ID NO: 28; CdtB/DNase^(Y174))ATGGAATGGGTAaagCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGAGGGAACAAGGTGCAGATATTGAGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTATGAGCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCCGACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCCCTGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTTAATGTTAAATCAGTTACGCTCACAAATTACAAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCTAACAAGCTGAAAGAAGC.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 28. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 28. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 28. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 28. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the chimera is encoded by the following aminoacid sequence:

(SEQ ID NO: 29; CdtB/DNase^(Y174))MEWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSREQGADIEMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRYEPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIRHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALPDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAALMLNQLRSQITSDHYPVEVMLKHHHHHHHM LEDPAANKLKEA.In another embodiment, the chimera is a homologue of SEQ ID NO: 29. Inanother embodiment, the chimera is a variant of SEQ ID NO: 29. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 29. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 29. Eachpossibility represents a separate embodiment of the present invention.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the chimera is encoded by the following nucleicacid sequence:

(SEQ ID NO: 30) ATGGAATGGGTAaagCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGAGGGAACAAGGTGCAGATATTGAGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTATGAGCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCCGACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCT AACAAGCTGAAAGAAGC.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 30. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 30. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 30. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 30. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the chimera is encoded by the following aminoacid sequence:

(SEQ ID NO: 31) MEWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSREQGADIEMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRYEPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIRHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKHHHHHHHMLEDPAA NKLKEA.In another embodiment, the chimera is a homologue of SEQ ID NO: 31. Inanother embodiment, the chimera is a variant of SEQ ID NO: 31. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 31. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 31. Eachpossibility represents a separate embodiment of the present invention.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the chimera is encoded by the following nucleicacid sequence:

(SEQ ID NO: 32) ATGGAATGGGTAAAGCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGGGAGAACAAGGTGCAGATATTTTGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTATGAGCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCGTACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCT AACAAGCTGAAAGAAGC.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 32. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 32. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 32. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 32. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the chimera is encoded by the following aminoacid sequence:

(SEQ ID NO: 33) MEWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADILMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRYEPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKHHHHHHHMLEDPAA NKLKEA.In another embodiment, the chimera is a homologue of SEQ ID NO: 33. Inanother embodiment, the chimera is a variant of SEQ ID NO: 33. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 33. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 33. Eachpossibility represents a separate embodiment of the present invention.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the chimera is encoded by the following nucleicacid sequence:

(SEQ ID NO: 34) ATGGAATGGGTAaagCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGAGGGAACAAGGTGCAGATATTGAGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTATGAGCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCCGACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTTAATGTTAAATCAGTTACGCTCACAAATTACAAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCT AACAAGCTGAAAGAAGC.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 34. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 34. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 34. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 34. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the chimera is encoded by the following aminoacid sequence:

(SEQ ID NO: 35) MEWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSREQGADIEMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRYEPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIRHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAALMLNQLRSQITSDHYPVEVMLKHHHHHHHMLEDPAA NKLKEA.In another embodiment, the chimera is a homologue of SEQ ID NO: 35. Inanother embodiment, the chimera is a variant of SEQ ID NO: 35. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 35. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 35. Eachpossibility represents a separate embodiment of the present invention.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the chimera is encoded by the following nucleicacid sequence:

(SEQ ID NO: 36) ATGGAATGGGTAaagCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGGGAGAACAAGGTGCAGATATTGAGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTATGAGCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCGTACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCT AACAAGCTGAAAGAAGC.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 36. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 36. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 36. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 36. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the chimera is encoded by the following aminoacid sequence:

(SEQ ID NO: 37) MEWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSGEQGADIEMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRYEPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKHHHHHHHMLEDPAA NKLKEA.In another embodiment, the chimera is a homologue of SEQ ID NO: 37. Inanother embodiment, the chimera is a variant of SEQ ID NO: 37. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 37. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 37. Eachpossibility represents a separate embodiment of the present invention.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the chimera is encoded by the following nucleicacid sequence:

(SEQ ID NO: 38) ATGGAATGGGTAaagCAATTAAATGTGGTTTTCTGTACGATGTTATTTAGCTTTTCAAGTTATGCTAACTTGAGTGATTTCAAAGTAGCAACTTGGAATCTGCAAGGTTCTTCAGCTGTAAATGAAAGTAAATGGAATATTAATGTGCGCCAATTATTATCGAGGGAACAAGGTGCAGATATTGAGATGGTACAAGAAGCGGGTTCATTACCAAGTTCGGCAGTAAGAACCTCACGAGTAATTCAACATGGGGGAACGCCAATTGAGGAATATACCTGGAATTTAGGTACTCGCTATGAGCCAAATATGGTCTATATTTATTATTCCCGTTTAGATGTTGGGGCAAACCGAGTGAACTTAGCTATCGTGTCACGTCGTCAAGCCGATGAAGCTTTTATCGTACATTCTGATTCTTCTGTGCTTCAATCTCGCCCGGCAGTAGGTATCCGCATTGGTACTGATGTATTTTTTACAGTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCAATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGCACCACCACCACCACCACCATATGCTCGAGGATCCGGCTGCT AACAAGCTGAAAGAAGC.In another embodiment, the nucleic acid encoding the chimera is ahomologue of SEQ ID NO: 38. In another embodiment, the nucleic acidencoding the chimera is a variant of SEQ ID NO: 38. In anotherembodiment, the nucleic acid encoding the chimera is an isoform of SEQID NO: 38. In another embodiment, the nucleic acid encoding the chimerais a fragment of SEQ ID NO: 38. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the chimera is encoded by the following aminoacid sequence:

(SEQ ID NO: 39) MEWVKQLNVVFCTMLFSFSSYANLSDFKVATWNLQGSSAVNESKWNINVRQLLSREQGADIEMVQEAGSLPSSAVRTSRVIQHGGTPIEEYTWNLGTRYEPNMVYIYYSRLDVGANRVNLAIVSRRQADEAFIVHSDSSVLQSRPAVGIRIGTDVFFTVHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAANGLSDQLAQAISDHYPVEVMLKHHHHHHHMLEDPAA NKLKEA.In another embodiment, the chimera is a homologue of SEQ ID NO: 39. Inanother embodiment, the chimera is a variant of SEQ ID NO: 39. Inanother embodiment, the chimera is an isoform of SEQ ID NO: 39. Inanother embodiment, the chimera is a fragment of SEQ ID NO: 39. Eachpossibility represents a separate embodiment of the present invention.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a deletion mutant comprises a single deletion. Inanother embodiment, a deletion mutant comprises a deletion of at least asingle nucleotide. In another embodiment, a deletion mutant comprises adeletion of at least a single amino acid. In another embodiment, adeletion mutant comprises a deletion of at least 2 amino acids. Inanother embodiment, a deletion mutant comprises a deletion of at least 3amino acids. In another embodiment, a deletion mutant comprises adeletion of at least 4 amino acids. In another embodiment, a deletionmutant comprises a deletion of at least 5 amino acids. In anotherembodiment, a deletion mutant comprises a deletion of at least 6 aminoacids. In another embodiment, a deletion mutant comprises a deletion ofat least 8 amino acids. In another embodiment, a deletion mutantcomprises a deletion of at least 10 amino acids.

In another embodiment, an insertion mutant comprises a single insertion.In another embodiment, an insertion mutant comprises an insertion of atleast a single nucleotide. In another embodiment, an insertion mutantcomprises an insertion of at least a single amino acid. In anotherembodiment, an insertion mutant comprises an insertion of at least 2amino acids. In another embodiment, an insertion mutant comprises aninsertion of at least 3 amino acids. In another embodiment, an insertionmutant comprises an insertion of at least 4 amino acids. In anotherembodiment, an insertion mutant comprises an insertion of at least 5amino acids. In another embodiment, an insertion mutant comprises aninsertion of at least 6 amino acids. In another embodiment, an insertionmutant comprises an insertion of at least 8 amino acids. In anotherembodiment, an insertion mutant comprises an insertion of at least 10amino acids.

In another embodiment, a substitution mutant comprises a singlesubstitution. In another embodiment, a substitution mutant comprises asubstitution of at least a single amino acid. In another embodiment, asubstitution mutant comprises a substitution of at least 2 amino acids.In another embodiment, a substitution mutant comprises a substitution ofat least 3 amino acids. In another embodiment, a substitution mutantcomprises a substitution of at least 4 amino acids. In anotherembodiment, a substitution mutant comprises a substitution of at least 5amino acids. In another embodiment, a substitution mutant comprises asubstitution of at least 6 amino acids. In another embodiment, asubstitution mutant comprises a substitution of at least 8 amino acids.In another embodiment, a substitution mutant comprises a substitution ofat least 10 amino acids.

In one embodiment, a mutation of the present invention comprises asubstitution of a sequence of DNase I with the large loop in the CdtBsequence, which in one embodiment, is present in residues L261-S272,which is predicted to bind to CdtA, in one embodiment. In oneembodiment, the DNase I fragment comprises a substitution of SEQ ID NO:58 for SEQ ID NO: 59.

In another embodiment, a mutated Cdt toxin or a mutated Cdt toxinfragment comprises an insertion mutation, a deletion mutation, asubstitution mutation, or any combination thereof. In anotherembodiment, a mutated CdtB toxin or a mutated CdtB toxin fragmentcomprises an insertion mutation, a deletion mutation, a substitutionmutation, or any combination thereof. In another embodiment, a mutatedDNase I or a mutated DNase I fragment comprises an insertion mutation, adeletion mutation, a substitution mutation, or any combination thereof.

In another embodiment, the sequence of a mutated chimera as providedherein may comprise the DNA sequence as set fourth in SEQ ID NOs: 28,30, 32, 34, 36, or 38. In another embodiment, the sequence of a mutatedchimera as provided herein may comprise the amino acid sequence as setfourth in SEQ ID NOs: 29, 31, 33, 35, 37, or 39.

In another embodiment, the chimera as provided herein is His-tagged. Inanother embodiment, the chimera as provided herein is His₆-tagged. Inanother embodiment, methods and compositions of the present inventionutilize a chimeric molecule, comprising a fusion of a recombinantchimeric polypeptide with a tag polypeptide that provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isplaced, in other embodiments, at the amino- or carboxyl-terminus of theprotein or in an internal location therein. The presence of suchepitope-tagged forms of the chimeric polypeptide is detected, in anotherembodiment, using an antibody against the tag polypeptide. In anotherembodiment, inclusion of the epitope tag enables the recombinantchimeric polypeptide to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. Various tag polypeptides and their respectiveantibodies are known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6): 547-553(1990)). Other tag polypeptides include the Flag-peptide (Hopp et al.,BioTechnology, 6: 1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science, 255: 192-194 (1992)); a tubulin epitope peptide (Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)); and the T7 gene 10protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87: 6393-6397 (1990)). Methods for constructing fusion proteins are wellknown in the art, and are described, for example, in LaRochelle et al.,J. Cell Biol., 139(2): 357-66 (1995); Heidaran et al., FASEB J., 9(1):140-5 (1995); Ashkenazi et al., Int. Rev. Immunol., 10(2-3): 219-27(1993) and Cheon et al., PNAS USA, 91(3): 989-93 (1994).

In another embodiment, provided herein is a chimera comprising a Cdtfragment in the amino terminus of the chimera and a DNase I fragment inthe carboxy terminus of the chimera. In another embodiment, providedherein is a chimera comprising a Cdt fragment in the amino terminus ofthe chimera and a homologue of a DNase I fragment in the carboxyterminus of the chimera. In another embodiment, provided herein is achimera comprising a homologue of a Cdt fragment in the amino terminusof the chimera and a DNase I in the carboxy terminus of the chimera. Inanother embodiment, provided herein is a chimera comprising a homologueof a Cdt fragment in the amino terminus of the chimera and a homologueof a DNase I fragment in the carboxy terminus of the chimera.

In another embodiment, provided herein is a chimera comprising a CdtBfragment in the amino terminus of the chimera and a DNase I fragment inthe carboxy terminus of the chimera. In another embodiment, providedherein is a chimera comprising a CdtB fragment in the amino terminus ofthe chimera and a homologue of a DNase I fragment in the carboxyterminus of the chimera. In another embodiment, provided herein is achimera comprising a homologue of a CdtB fragment in the amino terminusof the chimera and a DNase I in the carboxy terminus of the chimera. Inanother embodiment, provided herein is a chimera comprising a homologueof a CdtB fragment in the amino terminus of the chimera and a homologueof a DNase I fragment in the carboxy terminus of the chimera.

In another embodiment, provided herein is a recombinant polypeptidecomprising a chimera, wherein the recombinant polypeptide binds CdtC. Inanother embodiment, provided herein is a recombinant polypeptidecomprising a chimera, wherein the recombinant polypeptide binds CdtA. Inanother embodiment, provided herein is a recombinant polypeptidecomprising a chimera, wherein the recombinant polypeptide binds bothCdtA and CdtC. In another embodiment, provided herein is a recombinantpolypeptide consisting of a chimera, wherein the recombinant polypeptidebinds both CdtA and CdtC. In another embodiment, provided herein is arecombinant polypeptide consisting of a chimera and a leader peptide,wherein the recombinant polypeptide binds both CdtA and CdtC. In anotherembodiment, provided herein is a recombinant polypeptide consisting achimera and a signal peptide, wherein the recombinant polypeptide bindsboth CdtA and CdtC.

In another embodiment, provided herein is a recombinant polypeptide thatinhibits proliferation of a cell. In another embodiment, provided hereinis a recombinant polypeptide that inhibits proliferation of a eukaryoticcell. In another embodiment, provided herein is a recombinantpolypeptide that inhibits proliferation of a neoplastic cell. In anotherembodiment, provided herein is a recombinant polypeptide that inducescell cycle arrest. In another embodiment, provided herein is arecombinant polypeptide comprising a chimera, wherein the recombinantpolypeptide binds both CdtA and CdtC and induces cell cycle arrest. Inanother embodiment, provided herein is a recombinant polypeptidecomprising a chimera, wherein the recombinant polypeptide binds CdtC andinduces cell cycle arrest. In another embodiment, provided herein is arecombinant polypeptide comprising a chimera, wherein the recombinantpolypeptide binds CdtA and induces cell cycle arrest. In anotherembodiment, provided herein is a recombinant polypeptide comprising achimera, wherein the recombinant polypeptide binds both CdtA and CdtCand inhibits proliferation of a neoplastic cell. In another embodiment,provided herein is a recombinant polypeptide comprising a chimera,wherein the recombinant polypeptide binds CdtC and inhibitsproliferation of a neoplastic cell. In another embodiment, providedherein is a recombinant polypeptide comprising a chimera, wherein therecombinant polypeptide binds CdtA and inhibits proliferation of aneoplastic cell.

In another embodiment, provided herein is a recombinant polypeptidecomprising a chimera encoded by the DNA sequence of SEQ ID NO: 28,wherein the recombinant polypeptide binds both CdtA and CdtC andinhibits proliferation of a neoplastic cell. In another embodiment,provided herein is a recombinant polypeptide comprising a chimeraencoded by the amino acid sequence of SEQ ID NO: 29, wherein therecombinant polypeptide binds both CdtA and CdtC and inhibitsproliferation of a neoplastic cell.

In another embodiment, a chimera as described herein comprises a humanDNase I/CdtB protein comprising potent supercoiled DNA nicking activityand cell delivery and nuclear localization mechanisms.

In another embodiment provided herein is a a recombinant CdtApolypeptide comprising at least one of the mutations C149A and C178A,said polypeptide operably linked to a ligand that binds specifically toan antigen expressed on the surface of a cancerous epithelial cell type.In an exemplary embodiment, the antigen is prominin-1 or CD133.

A prominin-1 peptide or protein can be attached to heterologoussequences to form chimeric or fusion proteins. Such chimeric and fusionproteins comprise a peptide operatively linked to a heterologous proteinhaving an amino acid sequence not substantially homologous to thepeptide. “Operatively linked” indicates that the peptide and theheterologous protein are fused in-frame. The heterologous protein can befused to the N-terminus or C-terminus of the peptide.

In some uses, the fusion protein does not affect the activity of thepeptide or protein per se. For example, the fusion protein can include,but is not limited to, beta-galactosidase fusions, yeast two-hybrid GALfusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Suchfusion proteins, particularly poly-His fusions, can facilitate thepurification of recombinant prominin-1 proteins or peptides. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofa protein can be increased by using a heterologous signal sequence.

A chimeric or fusion prominin-1 protein or peptide can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different protein sequences are ligated together in-frame inaccordance with conventional techniques. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and re-amplified to generate a chimeric genesequence (see Ausubel et al., Current Protocols in Molecular Biology,1992-2006). Moreover, many expression vectors are commercially availablethat already encode a fusion moiety (e.g., a GST protein). Aprominin-1-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the prominin-1protein or peptide.

Variants of the prominin-1 protein can readily be identified/made usingmolecular techniques and the sequence information disclosed herein.Further, such variants can readily be distinguished from other peptidesbased on sequence and/or structural homology to the prominin-1 peptidesof the present invention. The degree of homology/identity present willbe based primarily on whether the peptide is a functional variant ornonfunctional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

Antibodies that selectively bind to the prominin-1 protein or peptidesof the present invention can be made using standard procedures known tothose of ordinary skills in the art. The term “antibody” is used in thebroadest sense, and specifically covers monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies, humanizedantibodies, and antibody fragments (e.g., Fab, F(ab′)₂, Fv andFv-containing binding proteins) so long as they exhibit the desiredbiological activity. Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules that lack antigen specificity. Antibodies can be of the IgG,IgE, IgM, IgD, and IgA class or subclass thereof (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2).

As used herein, antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains (referred to as an “intact” antibody).Each light chain is linked to a heavy chain by one covalent disulfidebond, while the number of disulfide linkages varies between the heavychains of different immunoglobulin isotypes. Each heavy and light chainalso has regularly spaced intrachain disulfide bridges. Each heavy chainhas at one end a variable domain (VH) followed by a number of constantdomains. Each light chain has a variable domain at one end (VL) and aconstant domain at its other end. The constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains. Chothia etal., J. Mol. Biol. 186:651-63 (1985); Novotny and Haber, Proc. Natl.Acad. Sci. USA 82:4592-4596 (1985).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of the environment in which it isproduced. Contaminant components of its production environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody willbe purified as measurable by at least three different methods: 1) togreater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight; 2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator; or 3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomasie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, the isolated antibody will be prepared by at leastone purification step.

An “antigenic region” or “antigenic determinant” or an “epitope”includes any protein determinant capable of specific binding to anantibody. This is the site on an antigen to which each distinct antibodymolecule binds. Epitopic determinants usually consist of active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as charge characteristics.

“Antibody specificity” refers to an antibody that has a stronger bindingaffinity for an antigen from a first subject species than it has for ahomologue of that antigen from a second subject species. Normally, theantibody “binds specifically” to a human antigen (i.e., has a bindingaffinity (Kd) value of no more than about 1×10⁷ M, preferably no morethan about 1×10⁻⁸ M and most preferably no more than about 1×10⁻⁹ M) buthas a binding affinity for a homologue of the antigen from a secondsubject species which is at least about 50 fold, or at least about 500fold, or at least about 1000 fold, weaker than its binding affinity forthe human antigen. The antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody (see, e.g., Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762; and 6,180,370).

The present invention provides an “antibody variant,” which refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues have been modified. Such variants necessarily haveless than 100% sequence identity or similarity with the amino acidsequence and have at least 75% amino acid sequence identity orsimilarity with the amino acid sequence of either the heavy or lightchain variable domain of the antibody, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Since the method of the invention appliesequally to both polypeptides and antibodies and fragments thereof, theseterms are sometimes employed interchangeably.

The term “antibody fragment” refers to a portion of a full-lengthantibody, including the antigen binding or variable region or theantigen-binding portion thereof. Examples of antibody fragments includeFab, Fab′, F(ab′)₂ and Fv fragments. Papain digestion of antibodiesproduces two identical antigen binding fragments, called the Fabfragment, each with a single antigen binding site, and a residual “Fc”fragment, so-called for its ability to crystallize readily. Pepsintreatment yields an F(ab′)2 fragment that has two antigen bindingfragments which are capable of crosslinking antigen, and a residualother fragment (which is termed pFc′). Additional antigen-bindingfragments can include diabodies, triabodies, tetrabodies, single-chainFv, single-chain Fv-Fc, a SMIP₅ and multispecific antibodies formed fromantibody fragments. As used herein, a “functional fragment” with respectto antibodies, refers to an Fv, F(ab), F(ab′)₂ or other antigen-bindingfragments comprising one or more CDRs that has the same antigen-bindingspecificity as an antibody.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three complementarity determining regions(“CDRs”) of each variable domain interact to define an antigen-bindingsite on the surface of the VH-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although typically at a lower affinity than the entire binding site.

The Fab fragment [also designated as F(ab)] also contains the constantdomain of the light chain and the first constant domain (CH1) of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains have a free thiol group. F(ab′) fragments areproduced by cleavage of the disulfide bond at the hinge cysteines of theF(ab′)2 pepsin digestion product. Additional chemical couplings ofantibody fragments are known to those of ordinary skill in the art.

A “single-chain Fv” or “scFv” antibody fragment contains V_(H) and V_(L)domains, wherein these domains are present in a single polypeptidechain. Typically, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the—scFv toform the desired structure for antigen binding. For a review of scFv,see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).A single chain Fv-Fc is an scFv linked to a Fc region.

A “diabody” is a small antibody fragment with two antigen-binding sites,which fragments comprise a variable heavy domain (V_(H)) connected to avariable light domain (V_(L)) in the same polypeptide chain. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. Diabodiesare described more fully in, for example, EP 0 404 097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).Triabodies, tetrabodies and other antigen-binding antibody fragmentshave been described by Hollinger and Hudson, 2005, Nature Biotechnology23:1126.

A “small modular immunopharmaceutical” or “SMIP” is a single-chainpolypeptide including a binding domain (i.e., an scFv or an antigenbinding portion of na antibody), a hinge region and an effector domain(e.g., an antibody Fc region or a portion thereof). SMIPs are describedin Published U.S. Patent Application No. 2005-0238646.

The present invention further provides monoclonal antibodies, polyclonalantibodies as well as chimeric and humanized antibodies, andantigen-binding fragments thereof to prominin-1 Tn general, to generateantibodies, an isolated peptide is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit or mouse.The full-length prominin-1 protein, or an antigenic peptide fragment ora fusion protein thereof, can be used as an immunogen. Particularlyimportant fragments are those covering functional domains, such as theextracellular domain or a portion thereof. Many methods are known forgenerating and/or identifying antibodies to a given target peptide.Several such methods are described by Harlow, Antibodies, Cold SpringHarbor Press (1989); Harlow and Lane, Using Antibodies, Cold SpringHarbor Press (1998); Lane, R. D., 1985, J. Immunol. Meth. 81:223-228;Kubitz et al., 1996, J. Indust. Microbiol. Biotech. 19:71-76; and Berryet al., 2003, Hybridoma and Hybridomics 22 (I): 23-31.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that substantially homogenous antibodies can be producedby a hybridoma culture which is uncontaminated by other immunoglobulinsor antibodies. The modifier “monoclonal” antibody indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein, Nature 256: 495-497 (1975) or may be made byrecombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. Themonoclonal antibodies for use with the present invention may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222: 581-597 (1991).

“Humanized” forms of non-human (e.g., murine or rabbit) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (a recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (a donor antibody) such asmouse, rat or rabbit having the desired, specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, a humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworkregion (FR) sequences. These modifications are made to further refineand optimize antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDRscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details, see: Jones et al.,Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988)and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen such as prominin-1protein, peptides or fragments thereof, and a carrier protein isprepared and an animal is immunized by the complex according to the samemanner as that described with respect to the above monoclonal antibodypreparation and the description in the Example. A serum or plasmacontaining the antibody against the protein is recovered from theimmunized animal and the antibody is separated and purified. The gammaglobulin fraction or the IgG antibodies can be obtained, for example, byuse of saturated ammonium sulfate or DEAE SEPHADEX, or other techniquesknown to those skilled in the art.

The antibody titer in the antiserum can be measured according to thesame manner as that described above with respect to the supernatant ofthe hybridoma culture. Separation and purification of the antibody canbe carried out according to the same separation and purification methodof antibody as that described with respect to the above monoclonalantibody and in the Example.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen. or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Antibodies, e.g., antibody variants, can be also made recombinantly.When using recombinant techniques, the antibody variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies that are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30minutes. Cell debris can be removed by centrifugation. Where theantibody variant is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore PELLICON ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight chain genes in E. coli is the subject of PCT publication numbersWO 901443, W0901443, and WO 9014424 and in Huse et al., 1989 Science246:1275-1281. The general recombinant methods are well known in theart.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and/or affinity chromatography, with affinity chromatographybeing the preferred purification technique.

An antibody against Promin-1 may be coupled (e.g., covalently bonded) toa Cdt polypeptide either directly or indirectly (e.g., via a linkergroup). A direct reaction between an antibody and a therapeutic agent ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one molecule may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide) onthe other molecule.

Alternatively, it may be desirable to couple a Cdt polypeptide and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the Pierce

Chemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g. U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), by protease cleavable linker (e.g., U.S. Pat. No.6,214,345), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody.

Regardless of the particular embodiment, conjugates with more than oneagent may be prepared in a variety of ways as described herein.

In another embodiment, provided herein is a method for inhibiting theproliferation of a neoplastic cell comprising the step of contacting acell with a recombinant polypeptide comprising a chimera as describedherein or a nucleic acid molecule encoding the same, thereby inhibitingthe proliferation of a neoplastic cell. In another embodiment, providedherein is a method for inhibiting the proliferation of a neoplastic cellcomprising the step of contacting a cell with a nucleic acid moleculeencoding a recombinant polypeptide comprising a chimera as describedherein comprising the step of administering a DNA vector comprising saidnucleic acid molecule. In one embodiment, methods of contacting a cellwith a nucleic acid molecule by administering a DNA vector are known toone of skill in the art.

In another embodiment, the vector consists of a DNA sequence encoding arecombinant polypeptide comprising a chimera and a larger sequence thatserves of the “backbone” of the vector. In another embodiment, thevector consists of a DNA sequence encoding a recombinant polypeptideconsisting of a chimera and a larger sequence that serves of the“backbone” of the vector. In another embodiment, the vector multipliesin the target cell. In another embodiment, the vector is expressed inthe target cell, thus expressing a recombinant polypeptide such as thosedescribed herein. In another embodiment, the vector is an expressionvector.

In one embodiment, the formulations and methods of the instant inventioncomprise a nucleic acid sequence operably linked to one or moreregulatory sequences. In one embodiment, a nucleic acid moleculeintroduced into a cell is in a form suitable for expression in the cellof the gene product encoded by the nucleic acid. Accordingly, in oneembodiment, the nucleic acid molecule includes coding and regulatorysequences required for transcription of a gene (or portion thereof).When the gene product is a protein or peptide, the nucleic acid moleculeincludes coding and regulatory sequences required for translation of thenucleic acid molecule include promoters, enhancers, polyadenylationsignals, sequences necessary for transport of an encoded protein orpeptide.

In one embodiment, nucleotide sequences which regulate expression of agene product (e.g., promoter and enhancer sequences) are selected basedupon the type of cell in which the gene product is to be expressed andthe desired level of expression of the gene product. For example, apromoter known to confer cell-type specific expression of a gene linkedto the promoter can be used. A promoter specific for myoblast geneexpression can be linked to a gene of interest to confer muscle-specificexpression of that gene product. Muscle-specific regulatory elementswhich are known in the art include upstream regions from the dystrophingene (Klamut et al., (1989) Mol. Cell Biol. 9:2396), the creatine kinasegene (Buskin and Hauschka, (1989) Mol. Cell Biol. 9:2627) and thetroponin gene (Mar and Ordahl, (1988) Proc. Natl. Acad. Sci. USA.85:6404). Negative response elements in keratin genes mediatetranscriptional repression (Jho Sh et al, (2001). J. Biol Chem).Regulatory elements specific for other cell types are known in the art(e.g., the albumin enhancer for liver-specific expression; insulinregulatory elements for pancreatic islet cell-specific expression;various neural cell-specific regulatory elements, including neuraldystrophin, neural enolase and A4 amyloid promoters). Alternatively, aregulatory element which can direct constitutive expression of a gene ina variety of different cell types, such as a viral regulatory element,can be used. Examples of viral promoters commonly used to drive geneexpression include those derived from polyoma virus, Adenovirus 2,cytomegalovirus (CMV) and Simian Virus 40, and retroviral LTRs.Alternatively, a regulatory element which provides inducible expressionof a gene linked thereto can be used. The use of an inducible regulatoryelement (e.g., an inducible promoter) allows for modulation of theproduction of the gene product in the cell. Examples of potentiallyuseful inducible regulatory systems for use in eukaryotic cells includehormone-regulated elements (e.g., see Mader, S. and White, J. H. (1993)Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulatedelements (see, e.g., Spencer, D. M. et al (1993) Science 262:1019-1024)and ionizing radiation-regulated elements (e.g., see Manome, Y. Et al.(1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl.Acad. Sci. USA89:1014-10153). Additional tissue-specific or inducibleregulatory systems which may be developed can also be used in accordancewith the invention.

In one embodiment, a regulatory sequence of the instant invention maycomprise a CMV promoter, while in another embodiment, the regulatorysequence may comprise a CAG promoter. In one embodiment, a CAG promoteris a composite promoter that combines the human cytomegalovirusimmediate-early enhancer and a modified chicken beta-actin promoter andfirst intron. In one embodiment, a regulatory sequence may comprise asimian virus (SV)-40 polyadenylation sequence, which in one embodiment,is the mechanism by which most messenger RNA molecules are terminated attheir 3′ ends in eukaryotes. In one embodiment, the polyadenosine(poly-A) tail protects the mRNA molecule from exonucleases and isimportant for transcription termination, for export of the mRNA from thenucleus, and for translation. In another embodiment, a formulation ofthe present invention may comprise one or more regulatory sequences.

In another embodiment, the vector comprises a promoter sequence thatdrives expression of the gene encoding a recombinant polypeptidecomprising a chimera, as described herein. In another embodiment, thevector is a transcription vector.

In another embodiment, the promoter is a constitutively active promoter.In another embodiment, the promoter is an inducible promoter.

In another embodiment, the vector is a plasmid. In another embodiment,the vector is a viral vector. In another embodiment, the vectorcomprises an origin of replication. In another embodiment, the vectorcomprises a “multiple cloning site”. In another embodiment, the vectoris a bacterial vector. In another embodiment, a bacterial vectorcomprising a transgene encoding a recombinant polypeptide as describedherein induces the expression of the recombinant polypeptide

In another embodiment, the vector is a genetically-engineered virus. Inanother embodiment, the vector further comprises a helper virus orpackaging lines for large-scale transfection. In another embodiment, thevector is designed for permanent incorporation of the insert into a hostgenome.

In another embodiment, methods for of contacting a cell with a nucleicacid provided herein include “naked DNA” technology. In anotherembodiment, methods including “naked DNA” technology result intransiently expressed recombinant polypeptide.

This invention provides, in one embodiment, a recombinant polypeptidecomprising a chimera, wherein said chimera comprises a DNase I fragmentor a homologue thereof and a Cdt fragment or a homologue thereof.

In one embodiment, “protein” or “polypeptide” refers to an amino acidchain comprising multiple peptide subunits, and may, in one embodiment,include a full-length protein, oligopeptides, and fragments thereof,wherein the amino acid residues are linked by covalent peptide bonds. Inone embodiment, a protein described in the present invention maycomprise a polypeptide of the present invention. In one embodiment, aprotein is a multimeric structure. In one embodiment, a protein of thepresent invention is a holotoxin.

The term “native” or “native sequence” refers to a polypeptide havingthe same amino acid sequence as a polypeptide that occurs in nature. Apolypeptide is considered to be “native” in accordance with the presentinvention regardless of its mode of preparation. Thus, such nativesequence polypeptide can be isolated from nature or can be produced byrecombinant and/or synthetic means. The terms “native” and “nativesequence” specifically encompass naturally-occurring truncated orsecreted forms (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of a polypeptide.

As used herein in the specification and in the examples section whichfollows the term “peptide” includes native peptides (either degradationproducts, synthetically synthesized peptides or recombinant peptides)and peptidomimetics (typically, synthetically synthesized peptides),such as peptoids and semipeptoids which are peptide analogs, which mayhave, for example, modifications rendering the peptides more stablewhile in a body or more capable of penetrating into bacterial cells.Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Either naturally occurring amino acids or non-conventional or modifiedamino acids may be used in the compositions and methods of the presentinvention.

As used herein, the term “amino acid” refers to either the D or Lstereoisomer form of the amino acid, unless otherwise specificallydesignated. Also encompassed within the scope of this invention areequivalent proteins or equivalent peptides, e.g., having the biologicalactivity of purified wild type tumor suppressor protein. “Equivalentproteins” and “equivalent polypeptides” refer to compounds that departfrom the linear sequence of the naturally occurring proteins orpolypeptides, but which have amino acid substitutions that do not changeit's biologically activity. These equivalents can differ from the nativesequences by the replacement of one or more amino acids with relatedamino acids, for example, similarly charged amino acids, or thesubstitution or modification of side chains or functional groups.

In another embodiment, the present invention provides a recombinantpolynucleotide encoding a recombinant polypeptide comprising a chimera,wherein said chimera comprises a DNase I fragment or a homologue thereofand a Cdt fragment or a homologue thereof.

In one embodiment, the term “nucleic acid” or “polynucleotide” refers tooligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA) or mimetic thereof. The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotide. This term includes oligonucleotides composed ofnaturally occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In one embodiment, the term “nucleic acid” or “oligonucleotide” refersto a molecule, which may include, but is not limited to, prokaryoticsequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. The term also refers to sequences that include any of theknown base analogs of DNA and RNA.

The nucleic acids can be produced by any synthetic or recombinantprocess such as is well known in the art. Nucleic acids can further bemodified to alter biophysical or biological properties by means oftechniques known in the art. For example, the nucleic acid can bemodified to increase its stability against nucleases (e.g.,“end-capping”), or to modify its solubility, or binding affinity tocomplementary sequences. These nucleic acids may comprise the vector,the expression cassette, the promoter sequence, the gene of interest, orany combination thereof. In another embodiment, its lipophilicity may bemodified, which, in turn, will reflect changes in the systems employedfor its delivery, and in one embodiment, may further be influenced bywhether such sequences are desired for retention within, or permeationthrough the skin, or any of its layers. Such considerations mayinfluence any compound used in this invention, in the methods andsystems described.

The term “promoter” means a nucleotide sequence that, when operablylinked to a DNA sequence of interest, promotes transcription of that DNAsequence.

DNA according to the invention can also be chemically synthesized bymethods known in the art. For example, the DNA can be synthesizedchemically from the four nucleotides in whole or in part by methodsknown in the art. Such methods include those described in Caruthers(1985). DNA can also be synthesized by preparing overlappingdouble-stranded oligonucleotides, filling in the gaps, and ligating theends together (see, generally, Sambrook et al. (1989) and Glover et al.(1995)). DNA expressing functional homologues of the protein can beprepared from wild-type DNA by site-directed mutagenesis (see, forexample, Zoller et al. (1982); Zoller (1983); and Zoller (1984);McPherson (1991)). The DNA obtained can be amplified by methods known inthe art. One suitable method is the polymerase chain reaction (PCR)method described in Saiki et al. (1988), Mullis et al., U.S. Pat. No.4,683,195, and Sambrook et al. (1989).

Methods for modifying nucleic acids to achieve specific purposes aredisclosed in the art, for example, in Sambrook et al. (1989). Moreover,the nucleic acid sequences of the invention can include one or moreportions of nucleotide sequence that are non-coding for the protein ofinterest. Variations in DNA sequences, which are caused by pointmutations or by induced modifications (including insertion, deletion,and substitution) to enhance the activity, half-life or production ofthe polypeptides encoded thereby, are also encompassed in the invention.

The formulations of this invention may comprise nucleic acids, in oneembodiment, or in another embodiment, the methods of this invention mayinclude delivery of the same, wherein, in another embodiment, thenucleic acid is a part of a vector.

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art as described hereinbelow.

As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire wild typegene or sequence. Likewise, forms of nucleic acid sequences can havevariations as compared to wild type sequences, nevertheless encoding theprotein or peptide of interest, or fragments thereof, retaining wildtype function exhibiting the same biological effect, despite thesevariations. Each of these represents a separate embodiment of thispresent invention.

In another embodiment, the present invention provides a DNA vectorcomprising a recombinant polynucleotide encoding a recombinantpolypeptide comprising a chimera, wherein said chimera comprises a DNaseI fragment or a homologue thereof and a Cdt fragment or a homologuethereof.

In one embodiment, the term “vector” or “expression vector” refers to acarrier molecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. In one embodiment,the vector is a DNA vector. In one embodiment, the vector is a plasmid.In one embodiment, the nucleic acid molecules are transcribed into RNA,which in some cases are then translated into a protein, polypeptide, orpeptide. In one embodiment, expression vectors can contain a variety of“control sequences” which refer to nucleic acid sequences necessary forthe transcription and possibly translation of an operably linked codingsequence in a particular host cell. In another embodiment, a vectorfurther includes an origin of replication. In one embodiment the vectormay be a shuttle vector, which in one embodiment can propagate both inprokaryotic and eukaryotic cells, or in another embodiment, the vectormay be constructed to facilitate its integration within the genome of anorganism of choice. The vector, in other embodiments may be, forexample, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus oran artificial chromosome. In one embodiment, the vector is a viralvector, which in one embodiment may be a bacteriophage, mammalian virus,or plant virus. In one embodiment, a vector is a plasmid.

In another embodiment, provided herein is a method for treating aneoplastic disease in a subject comprising the step of administering toa subject a recombinant polypeptide as described herein or a nucleicacid encoding the same, thereby treating a neoplastic disease in asubject.

In another embodiment, provided herein is a method for inhibiting orsuppressing a neoplastic disease in a subject comprising the step ofadministering to a subject a recombinant polypeptide as described hereinor a nucleic acid encoding the same, thereby inhibiting or suppressing aneoplastic disease in a subject.

In another embodiment, provided herein is a method for reducing thesymptoms associated with a neoplastic disease in a subject comprisingthe step of administering to a subject the recombinant polypeptide asdescribed herein or a nucleic acid encoding the same, thereby reducingthe symptoms associated with a neoplastic disease in a subject.

In one embodiment, the present invention provides a method forinhibiting the proliferation of a neoplastic cell comprising the step ofcontacting said cell with a recombinant polypeptide comprising a chimeraor a nucleic acid molecule encoding the same, wherein said chimeracomprises a DNase I fragment or a homologue thereof and a Cdt fragmentor a homologue thereof, thereby inhibiting the proliferation of aneoplastic cell.

In one embodiment, the present invention provides a method for treatinga neoplastic disease in a subject comprising the step of administeringto said subject a recombinant polypeptide comprising a chimera or anucleic acid encoding the same, wherein said chimera comprises a DNase Ifragment or a homologue thereof and a Cdt fragment or a homologuethereof, thereby treating a neoplastic disease in a subject.

In one embodiment, the present invention provides a method forinhibiting or suppressing a neoplastic disease in a subject comprisingthe step of administering to said subject a recombinant polypeptidecomprising a chimera or a nucleic acid encoding the same, wherein saidchimera comprises a DNase I fragment or a homologue thereof and a Cdtfragment or a homologue thereof, thereby inhibiting or suppressing aneoplastic disease in a subject.

In one embodiment, the present invention provides a method for reducingthe symptoms associated with a neoplastic disease in a subjectcomprising the step of administering to said subject a recombinantpolypeptide comprising a chimera or a nucleic acid encoding the same,wherein said chimera comprises a DNase I fragment or a homologue thereofand a Cdt fragment or a homologue thereof, thereby reducing the symptomsassociated with a neoplastic disease in a subject.

In one embodiment, the present invention provides a method forinhibiting the proliferation of a neoplastic cell comprising the step ofcontacting said cell with a recombinant polypeptide comprising achimera, wherein said chimera comprises a DNase I fragment or ahomologue thereof and a Cdt fragment or a homologue thereof, therebyinhibiting the proliferation of a neoplastic cell.

In one embodiment, the present invention provides a method for treatinga neoplastic disease in a subject comprising the step of administeringto said subject a recombinant polypeptide comprising a chimera, whereinsaid chimera comprises a DNase I fragment or a homologue thereof and aCdt fragment or a homologue thereof, thereby treating a neoplasticdisease in a subject.

In one embodiment, the present invention provides a method forinhibiting or suppressing a neoplastic disease in a subject comprisingthe step of administering to said subject a recombinant polypeptidecomprising a chimera, wherein said chimera comprises a DNase I fragmentor a homologue thereof and a Cdt fragment or a homologue thereof,thereby inhibiting or suppressing a neoplastic disease in a subject.

In one embodiment, the present invention provides a method for reducingthe symptoms associated with a neoplastic disease in a subjectcomprising the step of administering to said subject a recombinantpolypeptide comprising a chimera, wherein said chimera comprises a DNaseI fragment or a homologue thereof and a Cdt fragment or a homologuethereof, thereby reducing the symptoms associated with a neoplasticdisease in a subject.

In one embodiment, the present invention provides a method forinhibiting the proliferation of a neoplastic cell comprising the step ofcontacting said cell with a nucleic acid molecule encoding a recombinantpolypeptide comprising a chimera, wherein said chimera comprises a DNaseI fragment or a homologue thereof and a Cdt fragment or a homologuethereof, thereby inhibiting the proliferation of a neoplastic cell.

In one embodiment, the present invention provides a method for treatinga neoplastic disease in a subject comprising the step of administeringto said subject a nucleic acid encoding a recombinant polypeptidecomprising a chimera, wherein said chimera comprises a DNase I fragmentor a homologue thereof and a Cdt fragment or a homologue thereof,thereby treating a neoplastic disease in a subject.

In one embodiment, the present invention provides a method forinhibiting or suppressing a neoplastic disease in a subject comprisingthe step of administering to said subject a nucleic acid encoding arecombinant polypeptide comprising a chimera, wherein said chimeracomprises a DNase I fragment or a homologue thereof and a Cdt fragmentor a homologue thereof, thereby inhibiting or suppressing a neoplasticdisease in a subject.

In one embodiment, the present invention provides a method for reducingthe symptoms associated with a neoplastic disease in a subjectcomprising the step of administering to said subject a nucleic acidencoding a recombinant polypeptide comprising a chimera, wherein saidchimera comprises a DNase I fragment or a homologue thereof and a Cdtfragment or a homologue thereof, thereby reducing the symptomsassociated with a neoplastic disease in a subject.

In one embodiment, the present invention provides a use of a recombinantpolypeptide comprising a chimera or a nucleic acid molecule encoding thesame, wherein said chimera comprises a DNase I fragment or a homologuethereof and a Cdt fragment or a homologue thereof in the preparation ofa pharmaceutical composition for inhibiting the proliferation of aneoplastic cell.

In one embodiment, the present invention provides a use of a recombinantpolypeptide comprising a chimera or a nucleic acid molecule encoding thesame, wherein said chimera comprises a DNase I fragment or a homologuethereof and a Cdt fragment or a homologue thereof in the preparation ofa pharmaceutical composition for treating a neoplastic disease in asubject.

In one embodiment, the present invention provides a use of a recombinantpolypeptide comprising a chimera or a nucleic acid molecule encoding thesame, wherein said chimera comprises a DNase I fragment or a homologuethereof and a Cdt fragment or a homologue thereof in the preparation ofa pharmaceutical composition for inhibiting or suppressing a neoplasticdisease in a subject.

In one embodiment, the present invention provides a use of a recombinantpolypeptide comprising a chimera or a nucleic acid molecule encoding thesame, wherein said chimera comprises a DNase I fragment or a homologuethereof and a Cdt fragment or a homologue thereof in the preparation ofa pharmaceutical composition for reducing the symptoms associated with aneoplastic disease in a subject.

In one embodiment, a composition of the present invention is targeted toa rapidly proliferating cell, which in one embodiment, is anundifferentiated epithelial cell, and, in another embodiment, is alymphocyte. In one embodiment, immortalized epithelial-like cell linessuch as HeLa, KB, HEp-2 and GMSM-K (SV40 transformed) are particularlysensitive to Cdt, or in another embodiment, to Cdt-containing chimera ofthe present invention, or, in another embodiment, a CdtB-containingchimera of the present invention.

In one embodiment, a composition of the present invention may be used totreat, suppress, or inhibit a cancer of epitheloid origin, which in oneembodiment, is a carcinoma, which in one embodiment, is a squamous cellcarcinoma, adenocarcinoma, or transitional cell carcinoma, which in oneembodiment, is a lung, breast, ovarian, or colon cancer. In oneembodiment, the squamous cell carcinoma is oral squamous cell carcinoma.In one embodiment, breast cancer, prostate cancer, bladder cancer, braincancer and hepatic cancer are of epitheloid origin.

In another embodiment, basal cell carcinoma, gastrointestinal cancer,lip cancer, mouth cancer, esophageal cancer, small bowel cancer andstomach cancer, colon cancer, liver cancer, bladder cancer, pancreascancer, ovary cancer, cervical cancer, lung cancer, and skin cancer,such as squamous cell and basal cell cancers, renal cell carcinoma areof epitheloid origin. Other known cancers that effect epithelial cellsthroughout the body are known in the art.

In one embodiment, a neoplastic disease is a disease that involves theformation of a tumor or other abnormal tissue growth. In anotherembodiment, a neoplastic disease is a disease in which a neoplasm ispresent. In one embodiment, a neoplasm is an abnormal mass of tissue,the growth of which exceeds and is uncoordinated with that of the normaltissues, and persists in the same excessive manner after cessation ofthe stimulus which evoked the change. In one embodiment, a neoplasm doesnot form a tumor. In one embodiment, the neoplastic disease ismalignant. In another embodiment, the neoplastic disease ispre-malignant. In another embodiment, the neoplastic disease is benign.In one embodiment, the neoplastic disease is a sarcoma, lymphoma,leukemia, carcinoma, or multiple myeloma. in one embodiment, theneoplastic disease is diagnosed in soft tissue tumors of the head andneck, skin, salivary glands, thyroid gland, breast, skeletal system,maxilla, orbit or, temporal fossa, nervous system, lungs, pleura ormediastinum, esophagus or stomach, small intestine, large intestine,liver or gallbladder, pancreas, kidneys, adrenal, glands, or,ureters-carcinoma, urinary bladder-carcinoma, cancers of the femalegenital tract, prostate gland, testicles, or penis.

In another embodiment, the compositions and methods of the presentinvention may be used for reducing the symptoms associated withneoplastic disease. Such symptoms are dependent on the location of theneoplastic disease, and are known to a skilled artisan and may, in oneembodiment, include fevers, chills, night sweats, weight loss, loss ofappetite, fatigue, malaise, or a combination thereof. In one embodiment,lung tumors can cause coughing, shortness of breath, or chest painwhile, in one embodiment, tumors of the colon can cause weight loss,diarrhea, constipation and blood in the stool.

In one embodiment, the compositions and methods of the present inventionmay be used for treating, suppressing, or inhibiting tumor metastasis.In one embodiment, “metastasis” refers to the condition of spread ofcancer from the organ of origin to additional distal sites in thepatient.

In one embodiment, the compositions and methods of the present inventionmay be used for treating, suppressing, or inhibiting a “primary tumor”,which, in one embodiment, is a tumor appearing at a first site withinthe subject and can be distinguished from a “metastatic tumor” whichappears in the body of the subject at a remote site from the primarytumor. In one embodiment, a primary tumor is a solid tumor.

In one embodiment, the compositions and methods of the present inventionmay be used for treating, suppressing, or inhibiting wounds, ulcers,bronchitis, inflammatory conditions, herpes infection, or cysticfibrosis. In one embodiment, the compositions and methods of the presentinvention may be used for reducing the viscoelasticity of pulmonarysecretions (mucus). In one embodiment, the compositions and methods ofthe present invention may be used for treating, suppressing, orinhibiting pneumonia and cystic fibrosis (CF). See e.g., Lourenco, etal., Arch. Intern. Med. 142:2299-2308 (1982); Shak, et al., Proc. Nat.Acad. Sci. 87:9188-9192 (1990); Hubbard, et al., New Engl. J. Med.326:812-815 (1992); Fuchs, et al., New Engl. J. Med. 331:637-642 (1994);Bryson, et al., Drugs 48:894-906 (1994). In one embodiment, thecompositions and methods of the present invention may be used fortreating, suppressing, or inhibiting chronic bronchitis, asthmaticbronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, thecommon cold, in which, in one embodiment, mucus also contributes to itsmorbidity.

The examples provided herein demonstrate that functional chimeras of thecdtB and human DNase I genes may be obtained and strongly supports theclose relationship between the two gene products. The chimericcdtB-DNase I genes were expressed in E. coli and the gene productsexhibited nuclease activity, in vitro, comparable to that of CdtB. Thefact that these artificial prokaryotic/eukaryotic gene constructs wereexpressed by E. coli and satisfied very stringent requirements tomaintain a specific enzymatic activity provides compelling and novelevidence that the cdtB gene has evolved as an atypical divalentcation-dependent nuclease with remarkable similarities to mammalianDNase I.

In one embodiment, the successful construction of the chimeric genesindicates that there is significant potential to exploit the functionalrelationship between CdtB and human DNase I to genetically engineernovel therapeutic reagents that take advantage of the most desirablefeatures of each native protein. In one embodiment, DNase I has potentDNA-cutting/nicking activity. In one embodiment, CdtB can enter cellsand translocate to the cell nucleus. Thus, in one embodiment, the CdtBportion or portions of the chimera allows cell entry and translocationof the chimera and the DNase I portion or portions of the chimeraprovide potent DNA-cutting/nicking activity. In one embodiment, the CdtBportion or portions of the chimera confers cell type specificity to thechimera, which in one embodiment, is specificity for epitheloid cells.

In another embodiment, the compositions of the present invention furthercomprise a targeting molecule used to target the chimera to a particularcell type of interest. In one embodiment, the targeting molecule is anantibody to a specific polypeptide expressed by a tumor or to apolypeptide expressed by a particular cell type. In one embodiment,somatostatin, neurotensin, bombesin receptor binding molecules,monoclonal antibodies, Penetratines™, or glycoproteins, may be used astargeting molecules. Other targeting molecules are known in the art.

In another embodiment, provided herein is a method for inhibiting theproliferation of a cancerous epithelial cell type comprising:administering a recombinant CdtA polypeptide comprising at least one ofthe mutations C149A and C178A, said polypeptide operably linked to aligand that binds specifically to an antigen expressed on the surface ofa cancerous epithelial cell type.

In another embodiment, provided herein is a method for inhibiting theproliferation of a cancerous epithelial cell comprising: contacting saidcell with a recombinant CdtA polypeptide comprising at least one of themutations C149A and C178A, said polypeptide operably linked to a ligandthat binds specifically to an antigen expressed on the surface of saidcell.

In another embodiment, provided herein is a method for treating cancercomprising: administering a recombinant CdtA polypeptide comprising atleast one of the mutations C149A and C178A, said polypeptide operablylinked to a ligand that binds specifically to an antigen expressed onthe surface of a cancerous epithelial cell type.

In another embodiment, provided herein is a method for treating adisease associated with oral candidiasis comprising: administering arecombinant CdtA polypeptide comprising at least one of the mutationsC149A and C178A, said polypeptide operably linked to a ligand that bindsspecifically to an antigen expressed on the surface of a cancerousepithelial cell type.

In another embodiment, provided herein is a method for treating adisease associated with oral candidiasis comprising: administering atoxin composition specific to Candida albicans, said toxin compositioncomprising a recombinant CdtA polypeptide comprising at least one of themutations C149A and C178A, said polypeptide operably linked to a ligandthat binds specifically to an antigen expressed on the surface of acancerous epithelial cell type.

In one embodiment, “fragment” refers to a portion of a largerpolypeptide or polynucleotide. In one embodiment, a fragment retains oneor more particular functions as the larger molecule from which it wasderived. In one embodiment, a fragment may maintain a functional domain,which in one embodiment, is a nuclear localization signal (NLS), aglycosylation site, a cleavage site, a binding site, a DNA contact siteor residues, a G-actin binding site or residues, a metal binding site orresidues, a Sph chimera fusion site, a catalytic His, a Hi hydrogen-bondpair, or a combination thereof. In one embodiment, a fragment may beapproximately 50% of the length of the source polypeptide orpolynucleotide.

In another embodiment, a fragment may be approximately 90% of the lengthof the source polypeptide or polynucleotide. In another embodiment, afragment may be approximately 75% of the length of the sourcepolypeptide or polynucleotide. In another embodiment, a fragment may beapproximately 70% of the length of the source polypeptide orpolynucleotide. In another embodiment, a fragment may be approximately60% of the length of the source polypeptide or polynucleotide. Inanother embodiment, a fragment may be approximately 40% of the length ofthe source polypeptide or polynucleotide. In another embodiment, afragment may be approximately 30% of the length of the sourcepolypeptide or polynucleotide. In another embodiment, a fragment may beapproximately 25% of the length of the source polypeptide orpolynucleotide. In another embodiment, a fragment may be approximately10% of the length of the source polypeptide or polynucleotide. Inanother embodiment, a fragment may be approximately 5% of the length ofthe source polypeptide or polynucleotide.

In another embodiment, a fragment is approximately 150 amino acids. Inanother embodiment, a fragment is approximately 180 amino acids. Inanother embodiment, a fragment is approximately 200 amino acids. Inanother embodiment, a fragment is 100-200 amino acids. In anotherembodiment, a fragment is 125-175 amino acids. In another embodiment, afragment is 140-160 amino acids. In another embodiment, a fragment is50-150 amino acids. In another embodiment, a fragment is the equivalentnumber of nucleic acids required to encode an amino acid as described,as would be understood by a skilled artisan.

In one embodiment, “isoform” refers to a version of a molecule, forexample, a protein, with only slight differences to another isoform ofthe same protein. In one embodiment, isoforms may be produced fromdifferent but related genes, or in another embodiment, may arise fromthe same gene by alternative splicing. In another embodiment, isoformsare caused by single nucleotide polymorphisms.

In one embodiment, “variant” refers to an amino acid or nucleic acidsequence (or in other embodiments, an organism or tissue) that isdifferent from the majority of the population but is still sufficientlysimilar to the common mode to be considered to be one of them, forexample splice variants. In one embodiment, the variant may a sequenceconservative variant, while in another embodiment, the variant may be afunctional conservative variant. In one embodiment, a variant maycomprise an addition, deletion or substitution of 1 amino acid. In oneembodiment, a variant may comprise an addition, deletion, substitution,or combination thereof of 2 amino acids. In one embodiment, a variantmay comprise an addition, deletion or substitution, or combinationthereof of 3 amino acids. In one embodiment, a variant may comprise anaddition, deletion or substitution, or combination thereof of 4 aminoacids. In one embodiment, a variant may comprise an addition, deletionor substitution, or combination thereof of 5 amino acids. In oneembodiment, a variant may comprise an addition, deletion orsubstitution, or combination thereof of 7 amino acids. In oneembodiment, a variant may comprise an addition, deletion orsubstitution, or combination thereof of 10 amino acids. In oneembodiment, a variant may comprise an addition, deletion orsubstitution, or combination thereof of 2-15 amino acids. In oneembodiment, a variant may comprise an addition, deletion orsubstitution, or combination thereof of 3-20 amino acids. In oneembodiment, a variant may comprise an addition, deletion orsubstitution, or combination thereof of 4-25 amino acids.

In another embodiment, a chimeric polypeptide or isolated nucleic acidof the present invention is homologous to a sequence set forthhereinabove, either expressly or by reference to a GenBank entry. Theterms “homology,” “homologous,” etc, when in reference to any protein orpeptide, or any sequence, whether amino acid or nucleotide sequence,refer, in one embodiment, to a percentage of amino acid residues ornucleic acid residues, as appropriate, in the candidate sequence thatare identical with the residues of a corresponding native polypeptide ornucleic acid, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology, and not consideringany conservative substitutions as part of the sequence identity. Methodsand computer programs for the alignment are well known in the art.

Homology is, in another embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. For example,computer algorithm analysis of nucleic acid sequence homology caninclude the utilization of any number of software packages available,such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST EnhancedAlignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” refers to the identity between twosequences being greater than 70%. In another embodiment, “homology”refers to the identity between two sequences being greater than 72%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 75%. In another embodiment, “homology”refers to the identity between two sequences being greater than 78%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 80%. In another embodiment, “homology”refers to the identity between two sequences being greater than 82%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 83%. In another embodiment, “homology”refers to the identity between two sequences being greater than 85%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 87%. In another embodiment, “homology”refers to the identity between two sequences being greater than 88%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 90%. In another embodiment, “homology”refers to the identity between two sequences being greater than 92%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 93%. In another embodiment, “homology”refers to the identity between two sequences being greater than 95%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 96%. In another embodiment, “homology”refers to the identity between two sequences being greater than 97%. Inanother embodiment, “homology” refers to the identity between twosequences being greater than 98%. In another embodiment, “homology”refers to the identity between two sequences being greater than 99%. Inanother embodiment, “homology” refers to the identity between twosequences being 100%.

In another embodiment, homology is determined via determination ofcandidate sequence hybridization, methods of which are well described inthe art (See, for example, “Nucleic Acid Hybridization” Hames, B. D.,and Higgins S. J., Eds. (1985); Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y). In otherembodiments, methods of hybridization are carried out under moderate tostringent conditions, to the complement of a DNA encoding a nativecaspase peptide. Hybridization conditions being, for example, overnightincubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC(150 mM NaCl, 15 min trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA.

Homology for any amino acid or nucleic acid sequence listed herein isdetermined, in another embodiment, by methods well described in the art,including immunoblot analysis, or via computer algorithm analysis ofamino acid sequences, utilizing any of a number of software packagesavailable, via established methods. Some of these packages include theFASTA, BLAST, MPsrch or Scanps packages, and, in another embodiment,employ the use of the Smith and Waterman algorithms, and/or global/localor BLOCKS alignments for analysis, for example. Each method ofdetermining homology represents a separate embodiment of the presentinvention.

In some embodiments, any of the chimeric polypeptides or nucleic acidsof and for use in the methods of the present invention will comprise aDNase I fragment or a homologue thereof and a Cdt fragment or ahomologue thereof, or an isolated nucleic acid encoded said components,in any form or embodiment as described herein. In some embodiments, anyof the chimeric polypeptides or nucleic acids of and for use in themethods of the present invention will consist of a DNase I fragment or ahomologue thereof and a Cdt fragment or a homologue thereof, or anisolated nucleic acid encoded said components of the present invention,in any form or embodiment as described herein. In some embodiments, thechimeric polypeptides or nucleic acids of this invention will consistessentially of a DNase I fragment or a homologue thereof and a Cdtfragment or a homologue thereof, or an isolated nucleic acid encodedsaid components of the present invention, in any form or embodiment asdescribed herein. In some embodiments, the term “comprise” refers to theinclusion of other fragments of DNase I or Cdt, additional polypeptides,as well as inclusion of other proteins that may be known in the art. Insome embodiments, the term “consisting essentially of” refers to avaccine, which has a DNAse I polypeptide or fragment thereof and a Cdtfragment or a homologue thereof. However, other peptides may be includedthat are not involved directly in the utility of the chimericpolypeptide. In some embodiments, the term “consisting” refers to achimeric polypeptide having the specific DNAse I polypeptide or fragmentthereof and a Cdt fragment or a homologue thereof of the presentinvention, in any form or embodiment as described herein.

In one embodiment, a “chimeric” polypeptide or toxin is a protein ortoxin created through the joining of two or more nucleotides or geneswhich originally coded for separate proteins. In one embodiment,translation of this fusion polynucleotide results in a singlepolypeptide with functional properties derived from each of the originalproteins. In one embodiment, a chimeric polypeptide of the presentinvention comprises an IgE Fc or fragment thereof and a Cdt subunit.

PHARMACEUTICAL COMPOSITIONS

In another embodiment, the use of a recombinant polypeptide as describedhereinabove and/or its analog, derivative, isomer, metabolite,pharmaceutically acceptable salt, pharmaceutical product, hydrate,N-oxide, or combinations thereof for treating, preventing, suppressing,inhibiting or reducing the incidence of cancer.

Thus, in one embodiment, the methods of the present invention compriseadministering a recombinant polypeptide as described hereinabove. Inanother embodiment, the methods of the present invention compriseadministering a derivative of the recombinant polypeptide as describedhereinabove. In another embodiment, the methods of the present inventioncomprise administering an isomer of the recombinant polypeptide asdescribed hereinabove. In another embodiment, the methods of the presentinvention comprise administering a metabolite of the recombinantpolypeptide as described hereinabove. In another embodiment, the methodsof the present invention comprise administering a pharmaceuticallyacceptable salt of the recombinant polypeptide as described hereinabove.In another embodiment, the methods of the present invention compriseadministering a pharmaceutical product of the recombinant polypeptide asdescribed hereinabove. In another embodiment, the methods of the presentinvention comprise administering a hydrate of the recombinantpolypeptide as described hereinabove. In another embodiment, the methodsof the present invention comprise administering an N-oxide of therecombinant polypeptide as described hereinabove. In another embodiment,the methods of the present invention comprise administering any of acombination of an analog, derivative, isomer, metabolite,pharmaceutically acceptable salt, pharmaceutical product, hydrate orN-oxide of the recombinant polypeptide as described hereinabove.

As used herein, “pharmaceutical composition” means a “therapeuticallyeffective amount” of the active ingredient, i.e. the recombinantpolypeptide as described hereinabove, together with a pharmaceuticallyacceptable carrier or diluent. A “therapeutically effective amount”refers, in one embodiment, to that amount which provides a therapeuticeffect for a given condition and administration regimen.

The pharmaceutical compositions containing the recombinant polypeptideas described hereinabove can be administered to a subject by any methodknown to a person skilled in the art, such as parenterally,paracancerally, trans-mucosally, trans-dermally, intramuscularly,intravenously, intra-dermally, subcutaneously, intra-peritonealy,intra-ventricularly, intra-cranially, intra-vaginally orintra-tumorally.

In one embodiment, the pharmaceutical compositions are administeredorally, and are thus formulated in a form suitable for oraladministration, i.e. as a solid or a liquid preparation. Suitable solidoral formulations include tablets, capsules, pills, granules, pelletsand the like. Suitable liquid oral formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment of the present invention, the recombinant polypeptide asdescribed hereinabove is formulated in a capsule. In accordance withthis embodiment, the compositions of the present invention comprise, inaddition to the recombinant polypeptide as described hereinabove and theinert carrier or diluent, a hard gelating capsule.

Further, in another embodiment, the pharmaceutical compositions areadministered by intravenous, intraarterial, or intramuscular injectionof a liquid preparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously and are thus formulated in a form suitable for intravenousadministration. In another embodiment, the pharmaceutical compositionsare administered intraarterially and are thus formulated in a formsuitable for intraarterial administration. In another embodiment, thepharmaceutical compositions are administered intramuscularly and arethus formulated in a form suitable for intramuscular administration.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the recombinant polypeptides as describedhereinabove or their physiologically tolerated derivatives such assalts, esters, N-oxides, and the like are prepared and applied assolutions, suspensions, or emulsions in a physiologically acceptablediluent with or without a pharmaceutical carrier.

Further, in another embodiment, the pharmaceutical compositions areadministered as a suppository, for example a rectal suppository or aurethral suppository. Further, in another embodiment, the pharmaceuticalcompositions are administered by subcutaneous implantation of a pellet.In a further embodiment, the pellet provides for controlled release ofthe recombinant polypeptides as described hereinabove over a period oftime.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

As used herein “pharmaceutically acceptable carriers or diluents” arewell known to those skilled in the art. The carrier or diluent may be asolid carrier or diluent for solid formulations, a liquid carrier ordiluent for liquid formulations, or mixtures thereof.

Solid carriers/diluents include, but are not limited to, a gum, a starch(e.g. corn starch, pregeletanized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.microcrystalline cellulose), an acrylate (e.g. polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, and glycols such as propylene glycols orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,mineral oil, olive oil, sunflower oil, and fish-liver oil.

In addition, the compositions may further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the pharmaceutical compositions provided herein arecontrolled-release compositions, i.e. compositions in which therecombinant polypeptide as described hereinabove is released over aperiod of time after administration. Controlled- or sustained-releasecompositions include formulation in lipophilic depots (e.g. fatty acids,waxes, oils). In another embodiment, the composition is animmediate-release composition, i.e. a composition in which all of therecombinant polypeptide as described hereinabove is released immediatelyafter administration.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. For example, the agent may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment,polymeric materials can be used. In yet another embodiment, a controlledrelease system can be placed in proximity to the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984). Other controlled-release systems arediscussed in the review by Langer (Science 249:1527-1533 (1990).

The compositions may also include incorporation of the active materialinto or onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts.) Such compositions influence thephysical state, solubility, stability, rate of in vivo release, and rateof in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

Also comprehended by the invention are compounds modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline. The modified compounds are known toexhibit substantially longer half-lives in blood following intravenousinjection than do the corresponding unmodified compounds (Abuchowski etal., 1981; Newmark et al., 1982; and Katre et al., 1987). Suchmodifications may also increase the compound's solubility in aqueoussolution, eliminate aggregation, enhance the physical and chemicalstability of the compound, and greatly reduce the immunogenicity andreactivity of the compound. As a result, the desired in vivo biologicalactivity may be achieved by the administration of such polymer-compoundabducts less frequently or in lower doses than with the unmodifiedcompound.

The preparation of pharmaceutical compositions that contain an activecomponent, for example by mixing, granulating, or tablet-formingprocesses, is well understood in the art. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the recombinant polypeptide as described hereinabove ortheir physiologically tolerated derivatives such as salts, esters,N-oxides, and the like are mixed with additives customary for thispurpose, such as vehicles, stabilizers, or inert diluents, and convertedby customary methods into suitable forms for administration, such astablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions. For parenteral administration, therecombinant polypeptide as described hereinabove or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are converted into a solution, suspension, or emulsion, ifdesired with the substances customary and suitable for this purpose, forexample, solubilizers or other substances.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule), which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

In one embodiment, the term “treating” includes preventative as well asdisorder remitative treatment. As used herein, the terms “reducing”,“suppressing” and “inhibiting” have their commonly understood meaning oflessening or decreasing. As used herein, the term “progression” meansincreasing in scope or severity, advancing, growing or becoming worse.As used herein, the term “recurrence” means the return of a diseaseafter a remission.

In one embodiment, “treating” refers to either therapeutic treatment orprophylactic or preventative measures, wherein the object is to preventor lessen the targeted condition or disorder as described hereinabove.Thus, in one embodiment, treating may include directly affecting orcuring, suppressing, inhibiting, preventing, reducing the severity of,delaying the onset of, reducing symptoms associated with the disease,disorder or condition, or a combination thereof. Thus, in oneembodiment, “treating” refers inter alia to delaying progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In one embodiment, “preventing”refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

“Contacting,” in one embodiment, refers to directly contacting thetarget cell with a chimeric polypeptide of the present invention. Inanother embodiment, “contacting” refers to indirectly contacting thetarget cell with a chimeric polypeptide of the present invention. Thus,in one embodiment, methods of the present invention include methods inwhich the subject is contacted with a chimeric polypeptide which isbrought in contact with the target cell by diffusion or any other activetransport or passive transport process known in the art by whichcompounds circulate within the body.

In one embodiment, the methods of the present invention comprising thestep of administering a composition of the present invention to asubject in need. In one embodiment, “administering” refers to directlyintroducing into a subject by injection or other means a composition ofthe present invention. In another embodiment, “administering” refers tocontacting a cell with a composition of the present invention. In oneembodiment, the term “administering” refers to bringing a subject incontact with the recombinant polypeptide as described hereinabove. Asused herein, administration can be accomplished in vitro, i.e. in a testtube, or in vivo, i.e. in cells or tissues of living organisms, forexample humans. In one embodiment, the present invention encompassesadministering the compounds of the present invention to a subject. Inone embodiment, the compositions of the present invention areadministered chronically, while in another embodiment, they areadministered intermittently.

In one embodiment, “chronic” administration refers to administration ofthe agent(s) in a continuous mode as opposed to an acute mode, so as tomaintain a desired effect or level of agent(s) for an extended period oftime.

In one embodiment, “intermittent” administration is treatment that isnot consecutively done without interruption, but rather is periodic innature.

Administration “in combination with” or “in conjunction with” one ormore further therapeutic agents includes simultaneous (concurrent) andconsecutive administration in any order.

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic (including preventative) results. An effectiveamount can be administered in one or more administrations.

In one embodiment, the methods of the present invention compriseadministering a chimera as the sole active ingredient. In anotherembodiment, the present invention provides methods that compriseadministering a chimera in combination with one or more therapeuticagents.

EXPERIMENTAL DETAILS SECTION Materials and Methods

Construction of Chimeric cdtB Genes

Plasmids and primers used in this study are listed in Tables 1 and 2,respectively. All PCR and restriction digestion reactions were performedusing standard techniques. Restriction endonucleases were purchased fromNew England Biolabs (Beverly, Mass.) and PCR oligonucleotide primerswere obtained from Integrated DNA Technologies (IDT, Coralville, Iowa).Constructs were first selected in E. coli DH5α [supE44 ΔlacU169(φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] and transformedinto E. coli BL-21(DE3) for gene expression analysis and isolation ofthe gene product. Bacteria were grown in LB broth containing 75 μg/mlampicillin at 37′C with vigorous shaking. Plasmid DNA was isolated usingeither a QIAprep Miniprep (QIAGEN, Valencia, Calif.) or the WizardMiniprep Kit (Promega Corporation, Fitchburg, Wis.). All constructs andmutations were confirmed by DNA sequencing. Automated cycle sequencingreactions were conducted by the DNA Sequencing Facility at theUniversity of Pennsylvania Abramson Cancer Center using an AppliedBiosystems 96-capillary 3730XL sequencer with BigDye Taq FS Terminator V3.1.

The cdtB gene sequence used throughout these experiments was fromconstruct pJDB7 originally made in Cao et al., (2005) and based on thesequence deposited in GenBank (accession no. AF006830). The full lengthDNase I sequence (GenBank accession no. AC005203) was amplified fromplasmid GC-00001 (GeneCopoeia, Inc., Germantown, Md.) using the PCRprimers N-DNase-F and X-DNase-R. This amplicon was cloned, in frame, tothe His-tag in pETBlue2 (EMD Chemicals-Novagen, San Diego, Calif.) usingNcoI and XhoI restriction endonuclease cleavage sites. Portions of theDNA sequence from the resulting clone, pJD1, were used to construct thechimeras. To construct the DNase I/cdtB gene chimera the 5′-half of theDNase I gene sequence was obtained by digestion of pJD1 with NcoI andSphI, the 3′-half of the cdtB gene was obtained by digesting pJDB7 withSphI and BamHI and pET15b was cut with NcoI and BamHI. The three DNAfragments were ligated to obtain pETDB1 (Table 1). To construct thecdtB/DNase I gene chimera the PCR primers N-cdtB-F and S-cdtB-R wereused to amplify the 5′-half of cdtB using pJDB7 as the template DNA.pJD1 was digested with NcoI and SphI and the large DNA fragment wasligated to the pJDB7 amplicon after digestion with the same enzymes(clone pBDN1). The insert DNA fragment from pBDN1 was amplified usingthe PCR primers N-cdtB-F and NdeI-His and the amplicon was cloned intothe NcoI and NdeI sites of pET15b. The resulting plasmid, pETBDN2,contained the cdtB/DNase I chimeric gene, with codons for a His-tag atthe 3′-end, in the same vector background as the DNase I/cdtB genechimera.

TABLE 1 Plasmids used in this study Plasmid Features Source or referenceGC-C0001 human DNase I gene in GeneCopoeia pReceiver-B02 pJD1 DNase Igene containing 6 This study His codons at 3′-end in pETBlue2 pJDB7 cdtBgene containing 6 His Cao et al., 2005 codons at 3′-end in pET15bpMUT160cdtB pJDB7 with substitution This study H160A pDN1 human DNase Igene in This study pETBlue2 with 3′-His₆-tag pETDN1 human DNase I geneThis study containing 7 His codons at 3′-end in pET15b pBDN1 cdtB/DNaseI chimeric gene This study containing 6 His codons at 3′-end in pETBlue2pETBDN1 cdtB/DNase I hybrid gene in This study pET15b with 3′-His₆-tagpETBDN2 cdtB/DNase I chimeric gene This study containing 7 His codons at3′-end in pET15b pETDNB1 DNase I/cdtB chimeric gene This studycontaining 6 His codons at 3′-end in pET15b pETBDNmut5 pETBDN1containing 5 codon This study changes in the hybrid gene (Table 4)pETBDN2-1 pETBDN2 with substitutions This study S99Y and R100E pETBDN2-2pETBDN2-1 with This study substitution L62E pETBDN2-4 pETBDN2-3 withThis study substitution G55R pETBDN2-5 pETBDN2-4 with This studysubstitution V134R pETBDN2-5LL pETBDN2-5 with This study substitutionN265-I275 to L265-T275 pETBlue2 Cloning vector Novagen pET15b Cloningvector Novagen

Amino Acid Substitutions Mutants

Amino acid substitutions were made in the CdtB/DNase I hybrid protein bysite-directed mutagenesis as described previously (Cao et al., 2006).Synthetic oligonucleotide primer pairs were used to change S99Y andR100E (both in same primer), L62E, G55R and V134R. Briefly, mutant DNAstrands were made using PfuUltra DNA polymerase (Stratagene, La Jolla,Calif.) in PCR. Plasmid DNA from pETBDN2 was used as the first PCRtemplate. Additional mutations were then made sequentially using plasmidDNA from pETBDN2-1 to pETBDN2-5. Methylated parental DNA strands weredigested with DpnI and the intact mutated DNA strand was transformedinto E. coli TOP10 [F⁻ mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15AlacX74 recA1araD139Δ(ara-leu)7697 galU galK rpsL(str^(R)) endA1 nupG] chemicallycompetent cells (Invitrogen, Carlsbad, Calif.). The mutations wereconfirmed by sequencing of the plasmid insert DNA from a singletransformant.

The same strategy was used to change the active site H160 in CdtB-His₆to Ala. DNA from pJDB7 was used as the PCR template for primers H160forand H160rev. The mutated protein from this clone was used as a negativeactivity control in the bioassays.

The large loop domain was added to CdtB/DNase I using an inverse PCRmethod (Cao et al., 2004) with the Loop-F and Loop-R primers (Table 2)and the insert fragment from pETBDN2-5 (Table 1), containing thecdtB/DNase I nucleotide sequence, as the template DNA. Codons forLMLNQLRSQIT (SEQ ID NO: 58) replaced NGLSDQLAQAI (SEQ ID NO: 59) inpETBDN2-5LL.

TABLE 2 Primers used in this study SEQ ID Name Sequence* NO: N-DNase-F5′-AAATTACCATGGTGAGGGGAATGAAGC-3′ 40 X-DNase-R5′-TAATATTCTCGAGCTTCAGCATCACCT-3′ 41 N-cdtB-F5′-AAACGCGCCATGGAGTGGGTAAAGCAAT-3′ 42 S-cdtB-R5′-GGCCAAAGCATGCACTGTAAAA-3′ 43 NdeI-His 5′- 44ATTAACTTAATTACATATGGTGGTGGTGGTGGTGGTG- 3′ DNaseI-His 5′- 45TTTGGATCCGTGGTGGTGGTGGTGGTGCTTCAGCATCA C-3′ H160Afor 5′- 46ACTGATGTATTTTTTACAGTGGCTGCTTTGGCCACAGG TGG-3′ H160Arev 5′- 47CCACCTGTGGCCAAAGCAGCCACTGTAAAAAATACAT CAGT-3′ CdtB-DN-S99Y, 5′- 48R100E-F GAGGAATATACCTGGAATTTAGGTACTCGCTATGAGC CAAATATGGTCTATATTTA-TT-3′CdtB-DN-S99Y, 5′- 49 R100E-R AATAAATATAGACCATATTTGGCTCATAGCGAGTACCTAAATTCCAGGTATATTC-CTC-3′ CdtB-DN-L62E-F 5′- 50AATTATTATCGCGAGAACAAGGTGCAGATATTGAGAT GGTACAAGAAGC-3′ CdtB-DN-L62E-R 5′-51 GCTTCTTGTACCATCTCAATATCTGCACCTTGTTCTCG CGATAATAATT-3′ CdtB-DN-G55R-F5-AATGTGCGCCAATTATTATCGAGGGAACAAGGTGC- 52 3′ CdtB-DN-G55R-R5′-GCACCTTGTTCCCTCGATAATAATTGGCGCACATT-3′ 53 CdtB-DN-V134R-F 5′ 54CAAGCCGATGAAGCTTTTATCCGACATTCTGATTCTTC TGTGCT-3′ CdtB-DN-V134R-R 5′ 55AGCACAGAAGAATCAGAATGTCGGATAAAAGCTTCA TCGGCTTG-3′ Loop-F5′-CGCTCACAAATTACAAGTGACCACTATCCAG-3′ 56 Loop-R5′-TAACTGATTTAACATTAAGGCAGCCTGGAAG-3′ 57 *Underlined bases show the NcoI(CCATGG), XhoI (CTCGAG). SphI (GCATGC), NdeI (CATATG) and BamHI (GGATCC)restriction endonuclease cleavage sites. Bold type marks the changedcodon(s).

Isolation of Gene Products and Heterotoxin Reconstitution

Escherichia coli BL-21(DE3) [F⁻ ompT hsdS_(B) (r_(B) ⁻ m_(B) ⁻) gal dcm(DE3)] (Novagen) containing pJDA9 (wild-type CdtA-His₆), pJDC2(wild-type CdtC-His₆), pJDB7 (wild-type CdtB-His₆), pMUT160cdtB (activesite mutant), pETBDN2 (cdB-DNase I chimera), pETDNB1 (DNase I-cdBchimera) and pETBDN2-1 to pETBDN2-5(cdB-DNase I mutated chimera) wereused to isolate gene products by affinity chromatography onnickel-iminodiacetic acid columns (Novagen) as described previously (Caoet al., 2005). All of the gene products have a His₆₋₇ tag at thecarboxy-terminal end. The final protein preparations were dialyzed toremove urea, passed through 45 micron filters and quantified with theMicro BCA protein assay kit (Pierce, Rockville, Ill.). Purity wasassessed by analysis on 10-20% polyacrylamide gels. The presence of theHis-tag on all protein constructs was verified by western blotting usingAnti-His>>Tag Monoclonal Antibody (Novagen). Aliquots of the quantifiedprotein samples were stored at −70′C in a buffer containing 10 mMTris-HCl (pH 7), 100 mM NaCl, 5 mM MgCl₂ and 5 mM imidazole for use inthe various bioassays. Wild-type heterotoxin and heterotoxin containinghybrid and mutated hybrid proteins were reconstituted as describedpreviously (Mao & DiRienzo, 2002). Attempts to purify recombinant humanDNase I from either E. coli BL-21(DE3) (pJD1) or E. coli BL-21(DE3)(pETDN1) were unsuccessful most likely due to the poor expression of thehuman DNase I gene in E. coli as noted by Linardou et al. (2000).

In Vitro DNase Activity

Supercoiled DNA nicking activity was determined, with minormodifications, as described previously (Elwell & Dreyfus, 2000; Mao &DiRienzo, 2000). In a typical assay 1 μg of supercoiled pBluescript IISK(+) DNA (Sigma-Aldrich, St. Louis, Mo.) was incubated with 1 μg ofwild-type, mutant or hybrid CdtB protein in 25 mM HEPES (Sigma-Aldrich)(pH 7.0) containing 50 mM MgCl₂ at 37° C. for 1 h. DNase I (0.1 μg),from bovine pancreas (Sigma-Aldrich), was used in place of human DNaseI. Bovine DNase I is approximately 2.4-fold more active than the humanhomolog (Pan et al., 1998). Each form of DNA (supercoiled, relaxed,linear) was quantified and compared using ImageJ version 1.34(http://rsb.info.nih.gov/ij/) and digitized images of ethidiumbromide-stained gels. Protein and divalent cation concentrations werevaried in some kinetic and biochemical analyses.

Heat lability was compared by pretreating proteins at 100° C. for up to30 min prior to determining DNA nicking activity. Actin inhibition ofDNA nicking activity was performed essentially as described by Ulmer etal, (1996). Rabbit skeletal muscle G-actin (Sigma-Aldrich) wasde-polymerized at room temperature for 15 min in a buffer containing 25min HEPES (pH 7.5), 5 mM CaCl₂, 5 min MgCl₂, 0.1% BSA, 0.05% Tween 20and 0.5 min 2-mecaptoethanol. Five micrograms of the depolymerizedG-actin were preincubated with 1 μg of CdtB-His₆ or 0.1 μg of bovineDNase I for 60 min at 37° C. The treated proteins were then examined inthe nicking assay. Subunit protection of DNA nicking activity wasassessed as described previously (Ne{hacek over (s)}ić et al., 2004)except that incubation was performed at room temperature for 1 hour.

In Vivo CdtB Activity

Cell proliferation was measured with a colony-forming assay employingCHO cells [300 cells in 3 ml of medium per well (6-well plate) intriplicate] as described in Mao & DiRienzo (2002). The number ofcolonies per well was expressed as colony-forming units (CFU). A doseresponse curve for wild-type reconstituted heterotoxin has beenpublished (Cao et al., 2005). Cell cycle arrest was determined by flowcytometry of propidium iodide stained nuclei as described previously(Cao et al., 2005).

Binding Kinetics

Saturation kinetics were used to assess binding of the hybrid andmutated hybrid proteins to wild-type CdtA-His₆ and CdtC-His₆ in athyroglobulin ELISA (Cao et al., 2005). Wild-type CdtA-His₆ (4.0 μg) andCdtC-His₆ (3.5 μg) were added to thyroglobulin-coated wells. Hybrid ormutated hybrid protein (0-4 μg) was then added to triplicate wells.Bound protein was detected with a 1×10⁻⁶ dilution of anti-CdtB IgGrabbit antiserum and a 1×10⁻³ dilution of donkey anti-rabbitIgG-horseradish peroxidase conjugate (Amersham Pharmacia Biotech,Piscataway, N.J.) (Cao et al., 2008). The ability of the hybrid andmutated hybrid proteins to bind the other subunits was also determinedby stoichiometric binding in the thyroglobulin ELISA as describedpreviously (Cao et al., 2005). Wild-type CdtA-His₆ was prebound tothyroglobulin-coated 96-well microtiter plates as described above.Hybrid and mutated hybrid proteins (4.5 μg) and wild-type CdtC-His₆ (3.5μg) were added to triplicate wells. Total bound protein was detectedwith anti-HisTag monoclonal antibody and anti-mouse IgG horseradishperoxidase conjugate both at a 1×10⁻³ dilution. An absorbance ratio of3.0 is indicative of the binding of the hybrid or mutated hybrid proteinto the other two subunits. CdtB-His₆ and DNase I do not bind tothyroglobulin. ELISA plates were washed in a BioTek Model EL405 HTmicroplate washer (BioTek Instruments, Inc., Winooski, Vt.). Absorbancevalues were obtained with a BioTek Synergy 2 Multi-Detection MicroplateReader.

Differential dialysis was performed as described previously usingdialysis membrane with a molecular weight exclusion limit of 100 kDa(Spectrum Laboratories, Inc., Rancho Dominguez, Calif.) (Cao et al.,2008). Immunopositive bands on western blots were quantified withImageJ.

Computer Modeling

The European Molecular Biology Open Software Suite (EMBOSS release 3.0;http://emboss.sourceforge.net) (Rice et al., 2000) was used to obtainthe deduced amino acid sequences for cdtB-His₆ (from pJDB7), cdtB/DNaseI, DNase I/cdtB and human DNase I (from pETDN1) and to create thealignment using ClustalX 1.83. The crystal structures of the A.actinomycetemcomitans Y4 Cdt (Yamada et al., 2006) and bovine DNase Iwere modeled with UCSF Chimera 1.2197 (http://www.cgl.ucsf.edu/chimera/)(Pettersen et al., 2004). Coordinates were obtained from the ProteinData Bank (accession nos. 2F2F and 3DNI, respectively). Human DNase Ihas not been crystalized. CdtB/DNase I and DNase I/CdtB structures werepredicted using Modeller version 9v1 (http://salilab.org/modeller/)(Marti-Renom et al., 2000).

Polyclonal CdtB and CdtC Antisera

Subunit protein specific antisera for CdtB-His₆ and CdtCHis₆, used insome experiments, were made in rabbits (Cocalico Biologicals, Inc.,Reamstown, Pa.). IgG fractions were purified using a Montage AntibodyPurification Kit (Millipore, Billerica, Mass.). IgG titers were obtainedby ELISA using purified CdtB-His₆ and CdtC-His₆ as antigens.Cross-reactivity with all three Cdt proteins was assessed by westernblotting. Bound IgG was detected with a 1:3000 dilution of donkeyanti-rabbit IgG-horseradish peroxidase conjugate (Amersham PharmaciaBiotech).

Example 1 Using a CdtB/DNase I Hybrid Protein as a Therapeutic Agent

In spite of the phylogenetic relationship (FIG. 14) in CdtB/DNase, thereare key biological, compositional and structural differences between theCdtB and DNase I (Table 3). However, the similarities between theseproteins are strong enough to suggest that genetic modification ispossible to adapt them for potential use as growth inhibitors of cancercells.

TABLE 3 Comparison of physical and biochemical properties of CdtB andDNase I CdtB DNase I Property A. actinomycetemcomitans human bovineNumber of amino acids 283 282 261 Molecular weight (Da)^(a) 31,49431,434 29,183 pI^(b) 7.8 3.5-4.3 4.7-5.2 Disulfide no yes (C124-C127,yes (C102-C105, C196-C232) C174-C210) Carbohydrate modification^(c) noyes (N18 and N106) yes (N18) in vitro DNA nicking activity yes Yes yesActin inhibition no Yes yes Cations required for activity Mg⁺⁺ or Ca⁺⁺Mg⁺⁺ or Ca⁺⁺ Mg⁺⁺ or Ca⁺⁺ or Mn⁺⁺ or Mn⁺⁺ or Mn⁺⁺ Specific activity for100-1000 fold less 2.4 fold less — nicking dsDNA^(d) ^(a)Calculatedmolecular weight without carbohydrate moiety [additional 1.3 kDa percarbohydrate chain (NAG₂-MAN₅); 2 chains bovine, 1 chain human]^(b)Range due to heterogenity in glycosylation ^(c)Amino acid locationfrom processed proteins (minus signal sequence) ^(d)Relative to bovineDNase

DNase I cannot enter cells unaided. Even with some type of physical-,immuno- or ligand-based transport system the protein would not migrateto the cell nucleus. In contrast, CdtB appears to be taken up by cells(possibly with CdtC) via the Golgi complex and transported to theendoplasmic reticulum. However, this uptake occurs only after the Cdtbinds to the susceptible cell.

Initial binding may be to cholesterol-rich membrane patches (possiblylipid rafts). The CdtB travels to the nucleus by an unknown mechanismand gains access to the chromatin via a nuclear localization sequence.The CdtB sends the cells on a destructive pathway due to activation ofDNA damage checkpoint responses leading to growth arrest or apoptosis.These effects are most pronounced in rapidly proliferating cells such asundifferentiated epithelial cells and lymphocytes. The experimentsperformed revealed that immortalized epithelial-like cell lines such asHeLa, KB, HEp-2 and GMSM-K (SV40 transformed) are particularly sensitiveto the toxin. In contrast, oral primary fibroblast-like cell typesincluding human periodontal ligament fibroblasts (HPLF), human gingivalfibroblasts (HGF), cementoblasts, and osteoblasts are resistant to theDNA-damaging and cytotoxic effects of the Cdt.

The fact that CdtB is phylogenically related to human DNase I providesan additional advantage over typical prokaryotic-based recombinanttoxins. Furthermore, the DNA-damaging strategy (double-strand DNAbreaks) of the CdtB subunit of Cdt is likened to the effects of ionizingradiation. Targeting a potent DNA-damaging protein to the nucleus ofcancer cells is analogous to a form of localized radiation therapy.

Example 2 Kinetic Analysis of Recombinant CdtB-HIS₆ from A.actinomycetemcomitans and Bovine DNase I

The difference in specific activities between recombinant CdtB-His₆ andbovine DNase I is exemplified in FIG. 1. Purified recombinant A.actinomycetemcomitans Y4 CdtB from pJDB7 (FIG. 1 a), convertedsupercoiled [superhelical (S) circular, Form I] plasmid DNA to relaxed[(R) nicked circular, Form II] and linear [(L) Form III] forms with dosedependent kinetics in the standard DNA nicking assay (FIG. 1 b). Thisconversion was not affected by the location (amino- or carboxy-terminus)of the His₆-tag. One μg of recombinant CdtB-His₆ converted 95% of 1 μgof supercoiled plasmid DNA to relaxed or linear forms in 1 hour at 37°C. The substitution mutant CdtBH160A (from pMUT160cdtB in Table 1) hadno effect on supercoiled plasmid DNA confirming that recombinantCdtB-His₆ preparations were not contaminated with E. coli nucleases(data not shown). In comparison, 0.1 μg bovine DNase I converted 100% of1 μg of supercoiled plasmid DNA to relaxed form in 1 hour at 37° C.(FIG. 1 c). A ten-fold higher concentration of DNase I converted 98% of1 μg of supercoiled DNA to linear form and a 100-fold higherconcentration of the nuclease completely digested the DNA into smallfragments. Based on these results, the specific activity of recombinantCdtB-His₆ was estimated to be as much as 10³-fold lower than that ofbovine DNase I.

Maximum conversion of supercoiled DNA to relaxed and linear forms wasobtained with either 50 min MgCl₂, CaCl₂ or MnCl₂ (FIG. 2). Reactionscontaining CaCl₂ consistently failed to go to completion. CdtB-His₆nuclease activity was inhibited by MgCl₂, CaCl₂ and MnCl₂ when thecation concentrations were greater than 150-200 min. No conversion ofsupercoiled DNA was observed at any divalent cation concentration in theabsence of CdtB and various combinations of the cations (50 min of each)did not affect digestion patterns relative to those obtained with theindividual cations. A time course of digestion with 50 min MgCl₂ in thereaction established that 1 μg of CdtB-His₆ completely converted 1 μg ofsupercoiled DNA to relaxed and linear forms in 1 hour at 37° C. (datanot shown). Incubation times of 2 hours or greater generated more linearform DNA. Thus, CdtB-His₆ requires either Mg++, Ca++ or Mn++ at anindividual or combined concentration of 50 min for optimum activity.This is a 10-fold higher concentration than that required for optimumDNase I activity (Price, 1975). The standard in vitro DNase activityassay used in all subsequent experiments contained 50 min MgCl₂.

Cdt-B-His₆ retained greater than 90% of its DNA nicking activityfollowing incubation for 5 min in a boiling water bath (FIG. 3 A). Therewas a loss of virtually all enzymatic activity after heating for 10 min.In contrast, bovine DNase I DNA nicking activity was less than 50% ofthe control after 5 min of heating (FIG. 3B). The DNA nicking activityof both enzymes was significantly more heat stable than thedouble-strand cleavage activity.

CdtB-His₆ is not inhibited by G-actin (FIG. 4). These results confirmedthat both CdtB-His₆ and bovine DNase I have supercoiled DNA-nickingactivity and that the two enzymes can be distinguished by several keybiochemical properties (Table 3).

Example 3 Characterization of CdtB/DNase I and DNase I/CdtB ChimericGenes and Gene Products

Notable differences between the biochemical properties of CdtB and DNaseI suggested that a genetic strategy based on the analysis of theproducts of chimeric genes could be used to further examine functionalsimilarities and differences between the two proteins. This approach wasfacilitated by the presence of a unique SphI restriction endonucleasecleavage site at approximately the mid-point of both wild-type genesequences. Using each half of the CdtB-His₆ and human DNase I genesequences two genetic constructs, pETBDN2 and pETDNB1 containing thechimeric ORFs cdtB/DNase I and DNase I/cdtB, respectively (Table 1) weremade. Both ORFs were placed immediately downstream from a highlyefficient, inducible promoter and contained six or seven histidinecodons in frame at the 3′-end of each sequence.

Two active site catalytic histidines are conserved in CdtB from A.actinomycetemcomitans and bovine DNase I (FIG. 5A). A deduced amino acidsequence alignment showed that both hybrid gene products contain the twohistidines [residues H160/157 and H278/271 in CdtB/DNase I and DNaseI/CdtB, respectively] (FIG. 5C). Computer modeling indicated that onlythe CdtB/DNase I hybrid protein maintained a folded structure similar tothose of native CdtB and DNase I (FIG. 5B). However, both hybridproteins exhibited optimum in vitro supercoiled DNA nicking activity inthe presence of 50 min MgCl₂. The DNA nicking activity of CdtB/DNase Iwas not affected after incubation in a boiling water bath for 5 min butthere was a significant reduction in the DNA nicking activity of DNaseI/CdtB after the same treatment (FIG. 3). In contrast to bovine DNase I,G-actin had no effect on the ability of CdtB-His₆ and CdtB/DNase I toconvert supercoiled DNA to relaxed and linear forms. Conversely, actininhibited the DNA nicking activity of DNase I/CdtB (FIG. 4). The resultsof these assays demonstrated that the amino terminal portions of CdtBand DNase I carry the biochemical properties of thermostability andactin binding, respectively.

Example 4 Subunit Assembly of CdtB/DNase I and DNase I/CdtB HybridProteins

The ability of the hybrid proteins to form biologically activereconstituted heterotoxin was tested. Each hybrid protein was mixed withwild-type CdtA and CdtC in refolding buffer as described previously. CHOcell cultures were then treated with the reconstituted preparations.Neither hybrid protein yielded a biologically active heterotoxin asmeasured in the cell proliferation assay (FIG. 6A). A property specificto CdtB is the ability to bind the CdtA and CdtC subunits to form abiologically active heterotrimer. CdtB/DNase I but not DNase I/CdtBbound to CdtA-CdtC heterodimer at concentrations comparable to CdtB-His₆(FIG. 6 b). Both the CdtB-His₆ control and CdtB/DNase I hybrid proteinsreached saturation binding at approximately 3-4 μg per 4.0 μg ofCdtA-His₆ and 3.5 μg of CdtC-His₆. Consistent with these results,CdtB/DNase I but not DNase I/CdtB, formed a heterotrimer complex in thedifferential dialysis assay (FIG. 6 b; inset). In addition, CdtB/DNase Ibut not DNase I/CdtB bound stoichiometrically to CdtA and CdtC (FIG. 6c) and reconstituted heterotrimer preparations containing the CdtB/DNaseI hybrid protein failed to convert supercoiled DNA to linear and relaxedforms (FIG. 6 c; inset). Although these four independent assaysestablished that the CdtB/DNase I hybrid protein formed a heterotrimer,this complex failed to inhibit the proliferation and cell cycleprogression of CHO cells.

Computer modeling of the two hybrid proteins provided supportingevidence for reconciling the results of the proliferation inhibition(FIG. 6A) and thyroglobulin ELISA (FIG. 6B) assays. There are twostructural domains in CdtB, an α-helix labeled H1 (residues N41-S54) anda large loop (residues L261-S272), that are predicted to be importantfor the binding of this subunit to CdtC and CdtA, respectively (FIGS. 5and 7). A modeled ribbon structure of the CdtB/DNase I hybrid proteinshows that α-helix H1, located in the amino terminal half of CdtB, isstill present but the large loop, located in the carboxy-terminal halfof CdtB is missing. The presence of only the α-helix H1 suggests thatCdtB/DNase I is capable of binding only to CdtC. Addition of the largeloop CdtA-binding domain to CdtB/DNase I is discussed below. Incontrast, the ribbon model of the DNase I/CdtB hybrid protein containsonly the large loop structure and displays a significant change inoverall protein conformational relative to that of the native CdtB.

Example 5 Effect of Targeted Amino Acid Substitutions on the DNA Nickingand Subunit Binding Activities of CdtB/DNase I

Since the DNase I/CdtB hybrid protein failed to bind CdtA and CdtC (FIG.6), lacked a key domain for nuclear localization (FIG. 5C) and appearedto have an unfolded conformation in computer models (data not shown), itwas not studied further. Single amino acid substitutions weresequentially made in the CdtB/DNase I hybrid protein based on theresults from mutagenesis studies of the bovine and human forms of theDNase I gene (Pan et al., 1998). Substitutions G55R, S99Y and V134Radded DNA contact residues, L62E added a metal ion binding residue andR100E added a residue that hydrogen bonds to the catalytic H160 in DNaseI (FIG. 5C and Tables 1 and 4). Five hundred ng of the final mutatedhybrid gene product, CdtB/DNase I^(mut5)(clone pETBDN2-5), converted100% of 1 μg of supercoiled DNA to relaxed and linear forms understandard assay conditions resulting in an approximately 1.4-foldincrease in specific activity relative to CdtB-His₆ and CdtB/DNase I(FIG. 8 and inset B). CdtB/DNase I^(mut5) exhibited classicalfirst-order kinetics (inset A).

TABLE 4 Successive amino acid substitutions made in the CdtB/DNase Ihybrid protein. Position Amino acid (residue substitution number)^(a)Description of mutation

100 Add acidic residue that forms a hydrogen-bond pair with catalyticHis residue 160

55 Add DNA contact residue

99 Add DNA contact residue

134 Add DNA contact residue

62 Add metal ion binding residue ^(a)Based on the deduced amino acidsequence alignment of CdtB/DNase I and human DNase I (see FIG. 5).

Example 6 Cdt-Induced Cell Cycle Arrest of Human Cancer Cell Lines

As a prelude to determining the therapeutic efficacy of heterotoxinreconstituted with CdtB/DNase I^(mut5), experiments were initiated todetail the response of established human cancer cell lines to the nativeCdt. Four cancer cell lines derived from carcinomas of the tongue, nasalseptum and pharynx were purchased from the American Type CultureCollection (ATCC) (Table 5). HeLa cells were used as a transformed humancell control. The response of the cancer cell lines to that of normalprimary human gingival epithelial cells (HGEC strain AL-1) which havebeen recently isolated and cultured in our laboratory was performed.Each of the cell lines were treated with reconstituted recombinant A.actinomycetemcomitans Cdt for 24 hours and effects on the cell cyclewere assessed by flow cytometry as described previously. All fourCdt-treated cancer cell cultures exhibited a significant accumulation ofcells having a 4n DNA content indicative of a G₂ block in cell cycleprogression (Table 5). The portion of the cancer cell populations havinga 4n DNA content ranged from 49-70 percent compared to 44 and 45 percentfor HeLa and A1-1 cells, respectively. The accumulation of Cdt-treatedCAL-27 cells at the G₂/M interphase of the cell cycle is shown, comparedto controls, as an example in FIG. 9.

Thus, all of the cancer cell lines examined exhibited cell cycle arrestand on average appeared to be more sensitive to the Cdt then the HeLacells and normal primary HGEC. These cancer cell lines were used inexperiments described below to test the cytotoxic effects ofheterotrimer made with the final genetically modified hybrid protein.These cells are used to generate solid tumors in mice to test theefficacy of the hybrid protein.

TABLE 5 Cell cycle analysis of cancer cell lines treated withreconstituted Cdt Table 4. Cell cycle analysis of cancer cell linestreated with reconstituted Cdt Diploid G1 Diploid G2 Coefficient ATCC(2n) (4n) Diploid S of variance number Cell line^(a,b) Source DiseaseTreatment m (%) (%) (%) CRL-1624 SCC-4 tongue squamous cell none 73.987.42 18.59 5.33 carcinoma Cdt 17.90 63.52 18.58 4.61 CRL-2095 CAL-27tongue squamous cell none 74.35 2.04 23.61 5.57 carcinoma Cdt 39.2349.01 11.76 3.74 CCL-30 RPMI 2650 nasal squamous ceil none 73.87 8.7117.43 4.25 septum carcinoma Cdt 11.38 69.90 19.02 4.37 HTB-43 FaDupharynx squamous cell none 62.00 3.91 34.09 3.96 carcinoma Cdt 22.0156.06 21.93 4.46 CCL-2 HeLa cervix adenocarcinoma none 65.16 1.22 33.625.86 Cdt 39.19 43.58 17.23 4.16 — AL-1 human none none 43.57 24.42 32.018.43 gingiva Cdt 26.89 45.40 27.61 5.02 ^(a)All are adherent cells withepithelial cell morphology. ^(b)All cancer cell lines will form solidtumors in mice except HeLa cells.

Example 7 Modification of CdtB/DNase I^(mut5) to Improve Binding to CdtA

The genetically modified hybrid protein CdtB/DNase I^(mut5) (seeTable 1) had an increased specific activity, relative to CdtB andCdtB/DNase I (see FIG. 8), and formed a heterotrimer with wild-type CdtAand CdtC. However, this heterotrimer failed to inhibit the proliferationof cells or cause cell cycle arrest. As seen in the Cdt structuredepicted in FIG. 7, two regions in CdtB appear to be in contact withCdtA (large loop; L261-S272 in FIG. 5) and CdtC (α-helix H1; N41-S54 inFIG. 5). The large loop is not present in CdtB/DNase I^(mut5). TheCdtB/DNase I hybrid protein does not inhibit cell proliferation (seeFIG. 6A), yet appears to bind to the other subunits when CdtA isimmobilized on thyroglobulin or subjected to differential dialysis (FIG.6B). This result could be obtained if CdtB/DNase I^(mut5) binds in theheterotrimer only to CdtC instead of to both CdtA and CdtC thus formingan aberrant heterotrimer. To entice the CdtB/DNase I hybrid to form acorrectly assembled heterotoxin, the large loop region was added to theCdtB/DNase I^(mut5) hybrid protein by genetically substituting thesequence LMLNQLRSQIT (SEQ ID NO: 58, positions 261-271) for NGLSDQLAQAI(SEQ ID NO: 59, positions 265-275).

A computer model of CdtB/DNase I^(mut5) containing the restored largeloop sequence shows an increased distance (32.48 Å) between thisputative CdtA binding domain and the CdtC binding domain (α-helix H1)relative to that in CdtB (10.03 Å) (FIG. 10). It was hypothesized thatthis difference in conformation between CdtB and the hybrid protein mayaffect assembly of the heterotrimer. In a series of modeling exercises aproline residue was added at key theoretical folding regions in theCdtB/DNase I^(mut5) structure. One theoretical substitution, Y174P (FIG.5) reduced the estimated distance between the putative subunit bindingdomains to 15.38 Å. This proline was genetically added to the CdtB/DNaseI^(mut5) hybrid protein that contained the large loop modification.

The new mutated hybrid protein designated CdtB/DNase I^(Y174P) (SEQ IDNO: 29, 28), containing both the large loop and Y174P substitutions, wasused to make a heterotrimer with wild-type CdtA and CdtC. Like theprevious variations of the CdtB/DNase I hybrid proteins, CdtB/DNaseI^(Y174P) bound to CdtA and CdtC as measured in the thyroglobulin ELISA(FIG. 11). The stoichiometric increase in binding suggested that aheterotrimer was formed. Unlike previous versions of the hybrid protein,the heterotrimer formed with CdtB/DNase I^(Y174P) inhibitedproliferation of CHO cells (FIG. 12). To confirm that the new mutatedhybrid protein formed an active heterotoxin culture, HeLa cells and twoof the oral squamous cell carcinoma lines were treated with thereconstituted heterotrimer preparation. All three cell lines treatedwith heterotoxin made with CdtB/DNase I^(Y174P) exhibited a significantaccumulation of cells having a 4n DNA content indicative of a G₂ blockin cell cycle progression (Table 6). The portion of the cancer cellpopulations having a 4n DNA content ranged from 57-75 percent comparedto 39 percent for HeLa cells. The accumulation of CdtB/DNaseI^(Y174P)-treated cells at the G₂/M interphase of the cell cycle isshown, compared to controls, in FIG. 13.

TABLE 6 Cell cycle analysis of cancer cells treated with reconstitutedmutant heterotoxin Diploid Diploid Wild-type or G1 G2 DiploidCoefficient Cell mutant protein (2n) (4n) S of variance line inheterotrimer (%) (%) (%) (%) HeLa none 71.54 5.97 22.49 4.60 CdtB^(a)25.42 32.79 41.80 4.50 CdtB/DNase I^(Y174P) 30.06 38.75 31.19 4.47CCL-30 none 58.83 0.00 41.17 12.05 CdtB^(a) 18.47 54.09 27.44 5.32CdtB/DNase I^(Y174P) 19.91 56.73 23.37 6.10 CAL-27 none 75.38 2.63 21.994.68 CdtB^(a) 12.37 78.85 8.79 3.23 CdtB/DNase I^(Y174P) 11.51 74.5813.92 3.07 ^(a)Wild-type recombinant protein from E. coli BL-21(DE3)(pJDB7).

Example 8 Cell-Specific Targeting of a Microbial Genotoxin Results inSelective Inhibition of Cancer Stem Cells Materials and Methods

Epithelial Cells.

CAL-27, HeLa and AC133.1 (mouse B cell hybridoma; ATCC) were cultured inDMEM (Invitrogen). Caco-2, RPMI 2650 and FaDu were cultured in EMEMsupplemented with 0.1 min Non-Essential Amino Acids (Invitrogen-GIBCO).All cell lines were also supplemented with 10% FCS, except for Caco-2(20% FCS), and 0.1% Anti-Anti Antibiotic Antimycotic (Invitrogen-GIBCO).Primary human gingival epithelial cells (HGEC) were isolated fromgingival tissue removed as part of routine periodontal surgeriesconducted in the University of Pennsylvania School of Dental MedicineGraduate-Periodontics Clinic. The protocol for tissue procurementreceived Institutional Review Board approval and all donors wererequired to provide informed consent. Epithelial cells were isolated asdescribed previously. Since isolated cells typically reached senescenceby the third passage experiments requiring primary HGEC were repeatedusing cells isolated from gingival provided by different subjects.

HGEC isolated from a single explant culture were grown to 70% confluencein F12 medium containing 10% FCS and were transfected with 2 μg of pSG5TDNA, containing the SV40 large T-antigen gene. Effective TransfectionReagent (QIAGEN) was added according to the manufacturers instructions.Transfected cells were obtained by clonal dilution. Expression of theSV40 large T-antigen gene was confirmed by western blotting using ananti-SV40 large T- and small t-antigen-specific MAb (BD Pharmingen;1:200).

Cultures of a single immunopositive clone survived up to 22 passagesbefore reaching senescence. Intact cells were examined for the presenceof Ep-OAM by immunofluorescence staining

RNA Extraction and Real-Time PCR.

Total mRNA was isolated from cells using the Trizol reagent (Invitrogen)following the manufacturers recommendations. cDNA was prepared from 2 ugof total mRNA using oligo(dT) and the Superscript First-Strand SynthesisSystem for RT-PCR (Invitrogen). Real-time PCR reactions were preformedin an ABI7300 Real-Time PCR System using Power SYBR Green PCR Master Mix(Applied Biosystems). The cDNA-specific primers designed using thePrimer Express software (Applied Biosystems), were 5′GAGAAAGTGGCATCGTGCAA-3′ (forward) and 5′-TGCCAAACCAAAACAAATTCAA-3′(reverse). TATA-binding protein (TBP) mRNA served as normalizingcontrol. Forward and reverse primers were 5′-GGAGCTGTGATGTGAAGTTTCCTA-3′and 5′-CCAGGAAATAACTCTGGCTCATAAC-3′, respectively. A negative PCRcontrol without template cDNA was included in each assay.

Western Blotting.

CD133 was detected in whole cell lysates of the various SCC and primaryHGEC by western blotting using a standard procedure. Lysates of Caco-2and HeLa cells were used as positive and negative controls,respectively.

Site-Specific Mutagenesis.

The CdtA residue C178 was replaced with alanine by site directedmutagenesis using the primer pair5‘-AAAGTGTGTCACAAGGACGTGCAGTCACTTATAATCCTGTAAGTCC-3’ (forward) and5′-GGACTTACAGGATTATAAGTGACTGCACGTCCTTGTGACACACTT-TT-3¹(reverse). Theunderlined bases mark the alanine codon. Mutated DNA strands were madeusing PfuUltra DNA polymerase in PCR (Stratagene). Template DNA wasobtained from pMUTc149cdtA which contained a mutation resulting in theamino acid substitution C149A in CdtA. The methylated parental DNAstrands were digested with Dpn\ (New England Biolabs, Beverly, Mass.)prior to transformation of Escherichia coli TOP10 chemically competentcells (Invitrogen). The mutation was confirmed by DNA sequencing.Plasmid DNA having the confirmed sequence was isolated and transformedinto E. coli BL-21(DE3) competent cells (Novagen) to express the mutatedgene and for isolation of the gene product. The resulting doublecysteine mutant was designated CDTA^(c149A,C178A).

Protein Isolation and Toxin Reconstitution.

Recombinant clones E. coli BL-21(DE3) (pJDA9), E. coli BL-21(DE3)(pJDB7) and E. coli BL-21(DE3) (pJDC2) were used to prepare the threewild-type CdtA-His6, CdtB-HiS6 and CdtC-His6 proteins, respectively, byaffinity chromatography as described previously. All three proteins haveHis6 tags at the carboxy-terminal end. The same method was used toobtain the CdtA^(c149A, C178A)-His6 gene product. Protein purity wasassessed as described previously.

Wild-type heterotoxin (CdtABC) and heterotoxin containing theunconjugated and MAb-conjugated CdtA^(c149Ai C178A)-His6, protein werereconstituted in a refolding buffer as described previously.Heterotrimer formation was confirmed by a differential dialysistechnique using a membrane with a molecular weight exclusion limit of100 kDa (Spectrum Laboratories Inc.). Each reconstituted protein samplewas examined before and after dialysis on a western blot.

Binding Assay.

A previously characterized enzyme-linked immunosorbent assay for bindingof Cdt subunits to cultured cells (CELISA) was used to examine bindingkinetics of CdtA^(c149A) ^(—) ^(C178A)_His6. 96-well microtiter plateswere seeded with 1.5×10⁴ RPMI 2650 or FaDu cells/well in growth mediumand incubated for 48 h to allow the cells to attach and becomeconfluent. The cells were fixed with buffered 10% formalin followed bythe addition of increasing concentrations protein (0-2 μg of purifiedCdtA^(c149A, c178A)-HiS6 or cdtA-His₆) and 2% bovine serum albumin(BSA). After incubation for 1 h at room temperature bound protein wasfixed with a mixture of 2% formaldehyde and 2% glutaraldehyde anddetected with an anti-His>>Tag monoclonal antibody (Novagen).

Immunochemistry.

The IgG1 kappa fraction was purified from cultured AC133.1 mousehybridoma cells using the Montage™ Antibody Purification PROSEP-A Kit(Millipore). Antibody concentration was determined using theBeer-Lambert Law [IgG (mg/ml)=A280×0.72] and a titer was obtained usinga whole cell lysate of Caco-2 cells. The CdtA^(c149A,c178A)-His6-MAbconjugate was made using a modification of a procedure describedpreviously. The chemical crosslinker4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT;Pierce) was incubated with the purified IgG (1 mg SMPT/10 mg of IgG)overnight at 4° C. Excess SMPT was removed by dialysis.CdtA^(c149A,c178A)-His6 (0.5 mg) was added to the SMPT-MAb complex andthe preparation incubated for 48 h at 4° C. Unconjugated excess SMTP-MAbwas removed by passing the sample thorough a nickel-iminodiacetic acidcolumn (Novagen). Unconjugated CdtA^(c149A,c178A)-His6 was removed bydialysis of the sample against 20 min Tris-HCl (pH 7.9), 0.5 M NaCl and5 min imidazole. The final protein concentration was determined with theMicro BCA Protein Assay Kit (Pierce).

Cell Cycle Analysis.

Cell cycle arrest was determined by flow cytometry as describedpreviously. The various SCC cell lines, HGEC and Caco-2 cells were grownfor 24 h and then incubated for an additional 36 h with 10 ug/ml of oneof the following preparations: (i) reconstituted wild-type heterotoxin(CdtABC), (ii) heterotoxin made with MAb-conjugated mutated CdtA(CdtA^(c149A,c178A)BC-CD133MAb), heterotoxin made with unconjugatedmutated CdtA (CdtA^(c149A,C178A)BC) or (iv) purified AC 133.1 MAb IgG(CD133MAb). Input protein concentration was based on dose response dataobtained in published standardized assays. Untreated control cellcultures did not receive any additions. Propidium iodide stained nucleiwere prepared from 1×10⁶ cells from each culture as describedpreviously. Stained nuclei were examined with a FACSCalibur four-colordual-laser flow cytometer (BD Biosciences) at the Abramson Cancer CenterFlow Cytometry and Cell Sorting Resource Laboratory at University ofPennsylvania. The data from 30,000 events were analyzed with ModFit LT3.2 (Verity Software House).

In a separate set of experiments, cultures of the RPMI 2650 cell linewere incubated for 36 h with increasing concentrations (0-32 μg/ml) ofCdtA^(c149A,c178A)BC-CD133MAb.

Propidium iodide stained nuclei were prepared and examined by flowcytometry as described above to determine a dose response.

Immunofluorescence Staining.

Immunofluorescence microscopy was performed as described previously withminor modifications. Cells (1×10⁴ cells per well) were cultured ineight-well chamber slides for 48 h. Slides used for staining of CAL-27were coated with poly-L-lysine to promote cell attachment. Cells werefixed with 4% buffered formalin, blocked with 1% BSA and stained withmouse anti-human CD133/1 MAb (AC133, Miltenyi Biotec; 1:200) and AlexaFluor 488 goat anti-mouse IgG conjugate (Invitrogen/Molecular Probes®1:400). Cell nuclei were stained with DAPI. Slides were mounted usingProlong Anti-Fade (Promega Corporation) and viewed with a Nikon Eclipse80i fluorescence microscope. Digital images were recorded with SpotAdvanced 4.6 software (Diagnostic Instruments, Inc.).

For flow cytometry, 1×10⁶ cells, preblocked with 1% BSA, were incubatedwith either: (i) no antibody (unstained), (ii) an Alexa Fluor 488 goatanti-mouse IgG conjugate (Invitrogen) (Isotype ELIC stained), (iii)purified AC133.1 MAb IgG followed by the Alexa Fluor 488 conjugate (CD133MAb stained) or (iv) mouse anti-human CD133/1 MAb conjugated to PE.The data from 50,000 events were analyzed with FlowJo 8.8.6 (Tree Star,Inc.).

Computer Modeling.

The crystal structure of the A actinomycetemcomitans Y4 Cdt (38) wasmodeled with UCSF Chimera 1.3 (http://www.cgl.ucsf.edu/chimera/) usingProtein Data Bank accession number 2F2F coordinates. Disulfide bondswere predicted using the genomic disulfide analysis program (GDAP; 40;http://www.doe-mbi.ucla.edu/˜boconnor/GDAP/l)

Statistical Analyses.

Experiments were performed a minimum of three times on different daysunless otherwise noted. Data are presented as the mean values±SD.Standard deviations were for data sets obtained from independentexperiments. The Paired Student's f-test was used to compare differencesin data exhibiting normal distributions. P values<0.05 were consideredsignificant.

Results

Susceptibility of Primary HGEC and SCC Cell Lines to the Cdt.

HGEC were isolated from gingival tissue and immortalized with SV40.These cells had a characteristic epithelial morphology, were judged tobe free of fibroblasts by microscopic evaluation and contained thecharacteristic epithelial cell adhesion molecule (Ep-CAM; 13). Treatmentof primary (FIG. 15, first column) and. Immortalized. HGEC, in culture,with reconstituted recombinant A. actinomycetemcomitans Cdt (CdtABC)resulted in growth arrest at the G2/M interphase of the cell cycle.There was an approximate 50% increase in the number of cells with a 4nDNA content following exposure to 10 μg/ml of the toxin for 36 h. Whenhead and neck SCC cell lines CAL-27 (tongue), RPMI 2650 (nasal septum)and FaDu (pharynx) were exposed to the Cdt in culture, under the sameconditions they arrested at the G2/M interphase. From 47 to 61% of eachof the SCC cell populations had a 4n DNA content following exposure tothe toxin as shown for CAL-27 (FIG. 15, middle column). These resultswere similar to those obtained with a HeLa cell (adenocarcinoma of thecervix) control (FIG. 15, last column).

Gene Expression and Cell Surface Display of CD133.

Real-time (RT)-PCR showed that the RPMI 2650 cells expressed theprominin-1 gene at a level comparable to that of the colorectaladenocarcinoma Caco-2 positive control (FIG. 16A). The prominin-1 genewas also expressed by CAL-27 cells but at a level that was orders ofmagnitude, lower than that in RPMI 2650. Expression of the prominin-1gene was not detected in primary HGEC A prominin-1 sequence wasamplified from FaDu cDNA. However, the amount of amplicon made was notstatistically significant.

CD133 was observed in cell lysates of RPMI 2650 and Caco-2, on westernblots, using a CD133/1 (AC.133) mouse anti-human MAb (FIG. 16B) as wellas MAb IgG purified in our laboratory from hybridoma (AC133.1) culturemedium. CD133 was not detected in cell lysates of HGEC, FaDu or Cal-27cells. These results were in excellent agreement with those of theRT-PCR analysis.

To assess the distribution of CD133⁺ cells that displayed the antigen onthe cell surface in culture, immunolabeled SCC cell lines were examinedby fluorescence microscopy. CD133⁺ cells detected with CD133/1 (AC 133)mouse anti-human MAb were observed only in cultures of the RPMI 2650cell line (FIG. 17A). There was no detectable staining of CAL-27 cellswhich correlated with the expression data obtained by RT-PCR.Presentation of the CD133 antigen on the surface of RPMI 2650 cells wasconfirmed by flow cytometry (FIG. 17B). RPMI 2650 was the only SCC cellline that exhibited a significant increase in fluorescence intensitywhen evaluated with the mouse anti-human CD 133 MAb IgG (AC 133.1) and afluorescene-conjugated goat anti-mouse IgG reagent. This is indicated bythe rightward shift of the red peak relative to the positions of theblue and green peaks representing unstained cells and cells treated withthe fluorescene-conjugated second antibody (isotype FITC) alone,respectively. The positively stained RPMI 2650 cells exhibited a medianfluorescence (MF) of 57.5. The Caco-2 positive control cells, FaDu cellsand HGEC had MF values of 65.0, 5.1 and 12.7, respectively. Similarresults were obtained when the RPMI 2650 cells were stained using amouse anti-human CD 133 MAb conjugated to R-phycoerythrin (PE) (seeinset; MF=64.8). Taken together the results of the four CD 133 assaysestablished that the protein is a cell surface marker unique to certainSCC cell lines. Our findings are consistent with reports thatsubpopulations of tumor cell cultures contain CD133+ cells.

Targeting the Cdt to CD133⁺ Cells.

It is well established that the CdtA subunit binds the heterotrimer tohost cells via a surface exposed receptor. The objective was to inhibitthe formation of two predicted intrachain disulfides (C136/C149;C178/C197) in CdtA and to eliminate one of two surface exposed cysteines(FIG. 18A). Therefore, residues C149 and C178 were replaced withalanines. As shown in a CELISA, the double cysteine mutant,CdtA^(c149A), c178A lost the ability to bind to RPMI 2650 and FaDu cells(FIG. 18B). The difference in cell binding, relative to that of thewild-type CdtA, was statistically significant and occurred over inputprotein concentrations of >250 ng per well.

Differential dialysis showed that the mutated CdtA subunit formed aheterotrimer with wild-type CdtB and CdtC (FIG. 18C). Following dialysisall three Cdt subunits were recovered from the dialysis tubingcontaining the sample reconstituted with CdtA^(c149A,C178A).Heterotrimers, but not monomers and dimers, are retained by dialysistubing having a molecular weight exclusion limit of 100 kDa. Thedisulfide coupling agent SMPT was conjugated through an amide linkage tothe IgG isolated from the mouse B cell hybridoma AC 133.1. Thisconjugate was then coupled to CdtA^(c149A,C178A) through a disulfidelinkage using the remaining surface exposed C197.

Targeted Inhibition of SCC Cells. The CdtA^(c149A,c178A)-MAb conjugatewas combined with wild-type CdtB and CdtC in refolding buffer toreconstitute active heterotoxin. The various SCC and Caco-2 cell linesas well as primary and immortalized HGEC were treated in culture withthis reconstituted toxin. The cells were analyzed by flow cytometryafter 36 h of exposure to CdtA^(c149A, c178A)BC-CD133MAb (FIG. 5). Amongthe tumor cell lines, only Caco-2 and RPMI 2650 cells underwent arrestat the G2/M interphase of the cell cycle. On average, 19 and 15% ofthese cells populations (3.5×10⁶ cells), respectively, had a 4n DNAcontent after exposure to 10 μg/ml of toxin protein. Control culturestreated with either the mouse anti-human CD 133 IgG MAb (AC133.1) aloneor CdtA^(c149A,C178A)BC (reconstituted toxin containing the unconjugatedmutated CdtA) were not arrested at G2/M. Primary and immortalized HGECwere not affected by CdtA^(c149A,c78A)BC-CD133MAb even though greaterthan 90% of these cells arrested at G2/M when treated with the wild-typeCdt.

The RPMI 2650 cell line responded in a dose dependent fashion totreatment with CdtA^(c149A, c178A)BC-CD133MAb (FIG. 19, last panel).Response to this toxin was linear over the range of 0 to 16 μg/ml ofprotein (R-squared=0.99). Maximum inhibition was obtained withapproximately 16 μg/ml of protein. Higher concentrations of toxin showedsaturation kinetics indicating that cell cycle arrest was specific. Aportion (18%) of the total cell population had a 4n DNA contentfollowing exposure to the MAb-conjugated toxin. This response was abouthalf the number of cells that were arrested after exposure to thewild-type Cdt.

Intoxication experiments were routinely performed using cell culturesthat were 80-90% confluent. We found that younger cultures were moresensitive to both CdtABC and CdtA^(c149A,c178A)BC-CD133MAb. Whenreplicate 50% confluent cultures of RPMI2650 cells were untreated andtreated with 12 μg/ml of CdtABC or CdtA^(c149A,178A)BC-CD133MAb,8.5±0.7, 54.0±1.6 (P=0.001) and 26.4±0.6% (P=0.002) of cells,respectively, had a 4n DNA content. These results are indicative ofincreased accessibility of cells to the toxin.

Human epithelial cells and keratinocytes including HeLa, HEp-2, Caco-2,Vero, HaCat and Henle-407 are arrested at the G₂/M transition of thecell cycle when exposed to the Cdt from various bacterial genera. Wefound that the GMSM-K cell line was arrested at the S phase of the cellcycle when treated with recombinant A. actinomycetemcomitans Cdt. Thosestudies provided compelling evidence that epithelial cells may be invivo targets of the toxin. The susceptibility of primary humanepithelial cells to the Cdt had not been examined previously. In thisstudy we showed that the toxin induced human gingival epithelial cellsto arrest at the G2/M interphase. A similar result was obtained withthree established head and neck SCC lines. Obtaining these data was animportant first step in demonstrating that one can engineer thegenotoxin to selectively inhibit the proliferation of specific types orsubpopulations of cancerous epithelial cells.

The advantages of our method are twofold. First, the MAb was conjugatedto the cell binding subunit (CdtA) of the heterotrimer rather than tothe cytotoxic subunit (CdtB) leaving the active subunit of the toxin isunaltered. Second, application of the targeting strategy is unlimitedsince MAbs for other yet to be identified unique cancer cell surfacemarkers can readily be conjugated to the surface exposed cysteineresidue in CdtA^(c149A, c178A) to tightly control target cellspecificity. Our study serves as a “proof-of-concept” that the cellulartropism of the Cdt can be selectively altered and used to inhibit CSC inheterogeneous populations based on the expression of a unique cellsurface antigen. Our findings show that the molecule can be useful in aspecific antibody-based targeting of CSC that potentially outlines newprospects for more effective cancer treatments. Coupling of ananti-human CD 133 antibody with an agent capable of inducing cell deathvia a genotoxic mechanism may complement current cancer therapies byeliminating CSC responsible for tumor recurrence.

Example 9 Oral Candidasis Therapeutics Using a Microbial Genotoxin

Oral candidiasis has an exceptionally widespread effect on health sinceit is the most common fungal infection in human populations. In thegeneral population the disease is found more frequently in infants andolder individuals (denture wearers) and is prevalent inimmunocompromised patients that are undergoing cancer treatments,transplant procedures or are afflicted with HIV/AIDS.

Sufferers of oral candidiasis, which can include members of neonatal,pediatric, geriatric and immunosuppressed populations, are cared for bypediatric, geriatric and oral medicine dental practitioners. It isunknown, at this time if the cost of such treatment would be covered bymedical insurance. Antifungal agents, primarily nystatin and traizolessuch as fluconazole, are currently used for treatment. Accordingly,there exists a need for improved pharmaceutical compositions.

In one embodiment, the invention relates to a genetically andimmunochemically modified bacterial-produced cytotoxin. The cytolethaldistending toxin (Cdt) from the periodontal pathogen Aggregatibacteractinomycetemcomitans is a heterotrimer composed of CdtA, CdtB and CdtCsubunits. The active subunit, CdtB, displays a type Ideoxyribonuclease-like activity. While the primary function of CdtC isnot clear, we have shown that the CdtA subunit recognizes a specificreceptor on the surface of the target cell and that specific amino acidsin an aromatic amino acid motif are required for binding. The CdtAsubunit can be genetically engineered, without disrupting holotoxinassembly, to target the toxin specifically to pathogenic strains ofCandida albicans that are the etiological agents of oral candidiasis.Additionally, the invention relates to a construct having aCandida-specific Cdt that can be used in a topical formulation toinhibit the pathogen and to alleviate the clinical symptoms such aspain, dysgeusia and dysphagia. The product can be designed tospecifically target and inhibit the human fungal pathogen C. dlbicans.The product can be used in a topical formulation to treat oralcandidiasis. The product may also be useful for the treatment of otherforms of mucosal candidiasis, such as vaginal infections, that areamenable to topical application. The primary advantages of thispotential agent, over common antifungals, will be a high degree ofselective toxicity (a single species of yeast) and reduced likelihood ofthe development of resistant pathogenic C. albicans strains. There is anincreasingly rapid dissemination of resistances to routinely usedantifungal agents such as fluconazole.

Follow-on products can also be developed since any monoclonal antibodycan be attached to the genetically-modified CdtA protein. Furthermore,the invention relates to a product which is an anti-cancer agent thattargets the Prominin-1 antigen that has been found on the surface ofsome progenitor cancer cells.

It is a unique approach to adapt the Cdt to target a eukaryotic pathogeninstead of primary mammalian cells. Many types of mammalian eukaryoticcells, such as epithelial cells, lymphocytes and macrophages appear tobe the natural targets of the cytotoxin. However, there is evidence frompublished studies that yeast cells are sensitive to the DNA-damagingeffect of the Cdt. The stringent connection between DNA damage and cellcycle arrest is an essential safety feature in eukaryotic cells thatprevents cells with damaged DNA or chromatin from initiating celldivision. The cells cannot readily enhance the DNA repair pathway orcircumvent this checkpoint process by spontaneous mutation. Therefore,Candida can not be able to easily develop resistance to the cytotoxin.It is also a unique strategy to use the cell binding subunit of an A-Btype heterotoxin to make the cytotoxin cell-specific. The main advantageof this approach is that the biologically active component of theheterotoxin (CdtB subunit) remains unaltered.

Candida albicans-specific monoclonal antibodies can also be required tocreate the final product. Several C. albicans monocilonal antibodies arecommercially available (Millipore and Abeam). In addition, monoclonalantibodies that recognize antigens displayed on the surface of yeastcells are relatively easy to make and isolate. There are a number ofcompanies that specialize in monoclonal antibody production.

We have constructed mutants of CdtA that no longer bind to mammalianepithelial cells and have a single molecular surface exposed cysteinefor conjugation of the monoclonal antibodies. These mutated CdtAproteins form a heterotrimer complex, albeit inactive, with wild-typeCdtB and CdtC. Our data shows that a binding-deficientCdtA-anti-Prominin-1 conjugate recognizes Prominin-1 expressing celllines such as CaCo-2 as well as CAL-27, isolated from an oral squamouscell carcinoma.

Characterization of some of the binding-deficient and cysteinesubstitution mutants of CdtA has been published in: Cao, L., A. Volgina,C. M. Huang, J. Korostoff, and J. M. DiRienzo. 2005. Mol Microbiol58:1303-1321 and Cao, L., A. Volgina, J. Korostoff, and J. M. DiRienzo.2006. Infect Immun 74:4990-5002.

The key developmental steps in the process are: (1) To “knock out” theCdt receptor-binding domain in CdtA. (2) To mutate several trivialcysteine residues in CdtA to prevent possible intrachain disulfideformation. (3) To conjugate a linker-modified Candida monoclonalantibody to the mutated CdtA via the remaining molecular surface-exposedcysteine. (4) To reconstitute and test heterotoxin using the mutatedCdtA-Candida monoclonal antibody conjugated protein, wild-type CdtB andwild-type CdtC to confirm that this modified Cdt inhibits cells. (5) Todemonstrate that the Candida-specific Cdt reduces numbers of Candida andalleviates the pathology associated with oral candidiasis in a ratmodel.

A battery of assays, previously published by us, can be used to test theeffects of the modified heterotoxin on the growth of C. albicans inculture. An immortalized rat gingival epithelial cell line can also beused as a control to establish that the modified heterotoxin no longerinhibits the proliferation of epithelial cells.

Sprague-Dawley rats can be infected with C. albicans, using awell-characterized and published oral candidiasis rat model (Allen, C.M. 1994. Oral Surg Oral Med Oral Pathol 78:216-221 and Samaranayake, Y.H., and L. P. Samaranayake. 2001. Clin Microbiol Rev 14:398-429), andwill test the efficacy of the modified heterotoxin in vivo. At the endpoint of the experiments, tissue from euthanized rats can be processedto obtain colony forming units of the yeast and stained to assesshistological changes.

Example 11 Cytolethal Distending Toxin Induces Cell Damage in GingivalExplants

The cytolethal distending toxin (Cdt), expressed by the periodontalpathogen Aggregatibacter actinomycetemcomitans, inhibits theproliferation of sensitive cells by arresting the cell cycle at the G₂/Minterphase. This bacterium colonizes the subgingival microenvironment inclose proximity to sulcular and junctional epithelial cells. Thegingival epithelium is the first line of defense against periodontalpathogens and, when damaged, allows bacteria to collectively gain entryinto underlying connective tissue where the Cdt and other microbialproducts can affect infiltrating inflammatory cells leading to thedestruction of the attachment apparatus. Histological evaluation ofhealthy human gingival tissue, exposed to the Cdt for 18 hours ex vivo,revealed more rapid detachment of the keratinized epithelial cell layer,disruption of rete pegs, dissolution of tight-junctions and distensionof spinous and basal epithelial cells. Primary gingival epithelialcells, but not gingival fibroblasts, isolated from the same healthytissue were cell cycle arrested when treated with the toxin and examinedby flow cytometry. Our results: (i) show that the Cdt can make asignificant contribution to tissue destruction characteristic of theearly stages of periodontal disease and (ii) demonstrate that a gingivalexplant model can be used to further assess the virulence potential ofthis toxin.

Inventors of the instant application show that the epithelial layers ingingival explants obtained from periodontally healthy gingiva exhibitdetachment of the keratinized epithelial cell layer, disruption of retepegs, dissolution of tight-junctions and distension of spinous and basalepithelial cells when exposed to the Cdt. These data support thedevelopment of an ex vivo tissue model for assessing the effects of theCdt on gingival epithelial cells in situ. Establishing that the Cdt ofA. actinomycetemcomitans affects the integrity of the periodontiumjustifies targeting this toxin in therapeutic modalities designed toreduce the severity of periodontal diseases.

Materials and Methods Gingival Tissue and Primary Cells

Gingival tissue was collected during routine periodontal surgeriesconducted on healthy adults in the University of Pennsylvania School ofDental Medicine Graduate Periodontics Clinic. The protocol for tissueprocurement received Institutional Review Board approval and all donorsprovided informed consent. In some experiments epithelial cells (HGEC)and fibroblasts (HGF) were isolated from tissue samples as describedpreviously (Oda and Watson, 1990; Kanno et al., 2005). Cultures of HGECand HGF were grown in serum-free keratinocyte medium and DMEM mediumcontaining 10% FCS, respectively.

Protein Isolation and Toxin Reconstitution

Recombinant Cdt proteins were isolated by affinity chromatography asdescribed previously (Cao et al., 2005). Heterotrimers werereconstituted, in a refolding buffer, from wild-type subunits (Mao andDiRienzo, 2002) or CdtB^(H160A).

Histology and Immunostaining

Gingival tissue was immediately placed in F12 medium, supplemented with5% fetal bovine serum and 1% Antibiotic-Antimycotic (Invitrogen,Carlsbad, Calif.). The tissue was cut into 5 mm pieces and treated, inseparate experiments, with varying concentrations of reconstituted Cdtranging from 0 to 10 μg/ml. The toxin-treated samples were incubated at37° C., in an atmosphere containing 10% CO₂, for varying periods oftime, ranging from 0 to 36 h. Samples were then fixed in 4% bufferedformalin and processed for paraffin sections by the Tissue ProcessingService at the School of Dental Medicine. Sections from each explantwere routinely stained with hematoxylin and eosin. In some experimentsadditional sections were stained with pan-keratin Ab3 mouse monoclonalantibody (1:200; Lab Vision Products, Fremont, Calif.) and rabbitanti-CdtB polyclonal antibody (1:50,000; Cao et al., 2008) followed bygoat anti-mouse Alexa Fluor 488 and goat anti-rabbit IgG Alexa Fluor 594conjugate (1:400; Invitrogen). Cell nuclei were visualized with DAPI.The stained sections were viewed with a Nikon Eclipse 80i fluorescencemicroscope. Digital images were recorded with Spot Advanced 4.6 software(Diagnostic Instruments, Inc., Sterling Heights, Mich.). Experimentswere performed a minimum of three times using tissue obtained fromdifferent patients.

Isolated primary cells were grown on 8-well chamber slides and incubatedwith either 1:100 dilution of mouse anti-human Ep-Cam (EBA-1) antibody(Santa Cruz Biotechnology, Santa Cruz, Calif.) or 1:500 dilution ofanti-fibroblast CD90/Thy-1 antigen (Ab-1) antibody (Oncogene ResearchProducts, La Jolla, Calif.), followed by Alexa Fluor 488 goat anti-mouseIgG conjugate (1:400 dilution; Invitrogen) to assay for expression ofthe epithelial cell adhesion molecule (Ep-CAM) and CD90/Thy-1 antigen,respectively. Cell nuclei were stained with DAPI. The stained cells wereviewed with a laser scanning confocal microscope BioRad Radiance 2100(BioRad Laboratories, Hercules, Calif.).

Cell Cycle Analysis

Cell cultures were untreated or treated with 10 μg/ml of wild-type toxin(CdtABC) or toxin reconstituted with CdtB^(H160A) for 36 hours asdescribed previously (Cao et al., 2005). Cell cycle arrest wasdetermined by flow cytometry. Propidium iodide stained nuclei wereexamined on a FACSCalibur four-color dual-laser flow cytometer (BDBiosciences) at the Abramson Cancer Center Flow Cytometry and CellSorting Resource Laboratory of the University of Pennsylvania. Data from30,000 events were analyzed with ModFit LT 3.2 (Verity Software House,NH).

Results Stability of Gingival Tissue Explants

The general morphology of explants, maintained in tissue culture mediumfor up to 24 h, was examined to establish that gingival tissue removedduring periodontal surgery could be used to assess the effects of theCdt on the integrity of epithelial cells in situ. The gingivalepithelial layers (GE), rete pegs (RP) and connective tissue (CT) wereintact in hematoxylin-eosin stained sections from freshly excised tissue(FIG. 20A). The appearance of the epithelial cells and tight-junctions,detected by staining for pan-keratin expression, was normal. The onlyclearly evident change in appearance of the tissue was some mechanicalseparation of the keratinized surface layer by 18 h of incubation (FIG.20B). No further deterioration of the tissue was observed by 24 h (FIG.20C).

Effects of the Cdt on Gingival Tissue

Previous studies showed that a histidine in the catalytic site of theCdtB subunit is essential for the cell cycle arrest activity of the Cdt(Elwell et al., 2001). Although our mutant CdtB^(H160A) binds towild-type CdtA and CdtC, the reconstituted heterotrimer fails to inhibitthe proliferation of cells. No histological changes were observed ingingival tissue when treated, for up to 36 h, with 10 μg/ml ofreconstituted toxin containing the mutated CdtB subunit (CdtAB^(H160A)C)(FIG. 21A-C). The appearance of tissue treated with CdtAB^(H160A)C wasidentical to that observed with the untreated specimen (FIG. 21D).Tissue exposed to 10 μg/ml of wild-type toxin (CdtABC) for 18 hexhibited significant histologic alterations. There was more pronouncedpeeling of the keratinized surface layer, extensive disruption of theepithelial layers and marked the loss of structural integrity of therete pegs (FIG. 21E). In general, the epithelial layers appeared to beswollen and stained less intensely by hematoxylin-eosin. The epithelialcells were dramatically distended with an apparent loss of thetight-junctions (compare insets in FIGS. 21D and 21E). Tissue treatedfor 36 h with 5 μg/ml of Cdt exhibited the same effects (FIG. 20F). Theresults of dose response experiments indicated that tissue treated withas little as 2.5 μg/ml of Cdt for 24 h had less pronounced, but clearlydetectable, morphological changes.

Co-Localization of the Cdt and Epithelial Cells

Epithelial cells in Cdt-treated tissue were localized by stainingsections with anti-pan-keratin. When the same sections were co-stainedwith CdtB antibodies, the subunit was observed primarily in theepithelial layers (red fluorescence in FIG. 22A). Significantly lessCdtB was detected on the keratinized outer surface layer of the gingivalepithelium as well as in the underlying connective tissue. Similarresults were obtained when the tissue was treated with CdtAB^(H160A)C(FIG. 22B). However, in this case no change in tissue morphology wasobserved as noted by the appearance of more well delineated rete pegs.CdtB was not detected in untreated control tissue (FIG. 22C).

Effect of the Cdt on Primary Gingival Epithelial Cells and Fibroblasts

To further support the observations that the Cdt was primarily affectingthe epithelial and not connective tissue layers, we isolated primaryHGEC and HGF from the same type of tissue as that used for the ex vivoexperiments. The cultured primary HGEC had an epithelioid morphology,stained positive with a fluorescent-tagged Ep-CAM antibody and negativewith an antibody that recognizes CD90/Thy-1 (FIG. 23A). The cultured HGFexhibited a morphology typical of fibroblasts and bound the CD90/Thy-1(Ab-1) antibody. We previously showed that the Ab-1 marker is expressedin oral fibroblasts but not epithelial cells (Kang et al., 2005).

HGEC exposed to the Cdt were arrested at the G₂/M interphase of the cellcycle as indicated by a significant increase in the number of cells witha 4n DNA content (FIG. 23B). CdtAB^(H160A)C had no effect on the cellcycle. In contrast, there was no increase in the number of cells havinga 4n DNA content when the HGF were treated with the Cdt.

When we co-incubated healthy gingival tissue with reconstitutedrecombinant Cdt severe histological changes were observed by 18 hours ofexposure. The effects were observed well within the 48 hour window oftissue health. The swelling or distension of cells in the spinous andbasal layers of the tissue was a clear indication of Cdt activity. Thefact that the tissue was unaffected when exposed to toxin reconstitutedwith the biologically inactive CdtB^(H160A) subunit for up to 36 hours,localization of CdtB in the epithelial layers and cell cycle arrest ofCdt-treated primary HGEC, supplied additional supporting evidence thatthe observed tissue damage was directly related to the toxin. There wereno observed effects of the Cdt on cells in the connective tissue layer.This observation was supported by the data showing that very little CdtBwas present in the connective tissue and that primary HGF were notaffected by the Cdt.

In conclusion, gingival tissue exposed, in vitro, to the Cdt exhibitedsevere structural damage. The histologic changes were predominantlyconfined to the epithelial layers of the tissue making a strong case forthe role of the Cdt in the breakdown of the protective epithelialbarrier considered to be an early step in the initiation of periodontaldisease. The gingival explant model has significant potential forstudying the details of select interactions of the Cdt with gingivalepithelial cells in situ.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

1. A recombinant polypeptide comprising a chimera, wherein said chimeracomprises a DNase I fragment or a homologue thereof and a Cdt fragmentor a homologue thereof.
 2. The recombinant polypeptide of claim 1,wherein said Cdt fragment is a CdtB fragment.
 3. The recombinantpolypeptide of claim 2, wherein said CdtB fragment is as set forth inSEQ ID NO: 14 or a fragment thereof.
 4. The recombinant polypeptide ofclaim 2, wherein said CdtB fragment comprises a mutation.
 5. Therecombinant polypeptide of claim 4, wherein said mutation is G55R, L62E,S99Y, R100E, V134R, Y174P or their combination.
 6. The recombinantpolypeptide of claim 4, wherein said DNase I fragment comprises asubstitution of SEQ ID NO: 58 for SEQ ID NO:
 59. 7. The recombinantpolypeptide of claim 1, wherein said DNase I fragment is encoded by asequence as set forth in SEQ ID NO: 1-7 or a fragment thereof.
 8. Therecombinant polypeptide of claim 1, wherein said chimera is His₆-tagged.9. The recombinant polypeptide of claim 1, wherein said chimeracomprises (a) said CdtB fragment in the amino terminus of said chimeraand said DNase I fragment in the carboxy terminus of said chimera or (b)a homologue of a DNase I fragment in the carboxy terminus of saidchimera and a Cdt fragment in the amino terminus of said chimera or (c)a DNase I in the carboxy terminus of said chimera and a homologue of aCdt fragment in the amino terminus of said chimera or (d) a homologue ofa DNase I fragment in the carboxy terminus of said chimera and ahomologue of a Cdt fragment in the amino terminus of said chimera. 10.The recombinant polypeptide of claim 1, wherein the amino acid sequenceof said chimera is as set forth in SEQ ID NO: 24-27.
 11. The recombinantpolypeptide of claim 1, wherein said recombinant polypeptide binds bothCdtA and CdtC.
 12. The recombinant polypeptide of claim 1, wherein saidrecombinant polypeptide inhibits proliferation of a neoplastic cell. 13.The recombinant polypeptide of claim 1, wherein said recombinantpolypeptide inhibits proliferation of a neoplastic cell and binds bothCdtA and CdtC.
 14. The recombinant polypeptide of claim 1, wherein theamino acid sequence of said recombinant polypeptide is as set forth inSEQ ID NO:
 29. 15. A recombinant polynucleotide encoding the chimera ofclaim
 1. 16. The recombinant polynucleotide of claim 15, wherein thenucleic acid sequence encoding said chimera is as set forth in SEQ IDNO: 22-23.
 17. The recombinant polynucleotide of claim 15, wherein thenucleic acid sequence of said chimera is as set forth in SEQ ID NO: 28.18. A DNA vector comprising the recombinant polynucleotide of claim 15.19. A plasmid comprising the recombinant polynucleotide of claim
 15. 20.The DNA vector of claim 17, wherein said vector further comprises apromoter.
 21. The DNA vector of claim 19, wherein said promoter is aconstitutively active promoter.
 22. A method for inhibiting theproliferation of a neoplastic cell comprising the step of contactingsaid cell with a recombinant polypeptide according to claim 1 or anucleic acid molecule encoding the same. 23.-39. (canceled)
 40. A methodfor treating a neoplastic disease in a subject comprising the step ofadministering to said subject a recombinant polypeptide according toclaim 1 or a nucleic acid encoding the same. 41.-57. (canceled)
 58. Amethod for inhibiting or suppressing a neoplastic disease in a subjectcomprising the step of administering to said subject a recombinantpolypeptide according to claim 1 or a nucleic acid encoding the same.59.-75. (canceled)
 76. A method for reducing the symptoms associatedwith a neoplastic disease in a subject comprising the step ofadministering to said subject a recombinant polypeptide according toclaim 1 or a nucleic acid encoding the same. 77.-93. (canceled)
 94. Acomposition comprising: a recombinant CdtA polypeptide comprising atleast one of the mutations C149A and C178A, said polypeptide operablylinked to a ligand that binds specifically to an antigen expressed onthe surface of a cancerous epithelial cell type.
 95. (canceled) 96.(canceled)
 97. The composition of claim 94, wherein said cell type is aprogenitor stem cell.
 98. The composition of claim 94, wherein saidcancer is a squamous cell carcinoma.
 99. The composition of claim 94,wherein said mutant CdtA polypeptide encodes a genotoxin.
 100. Thecomposition of claim 94, wherein said mutant CdtA polypeptide is setforth in SEQ ID NO:
 9. 101. The composition of claim 94, wherein saidantigen is prominin-1 or CD133.
 102. The composition of claim 94,wherein said ligand is a protein that binds specifically to saidantigen.
 103. The composition of claim 94, wherein said ligand is anantibody that binds specifically to said antigen.
 104. The compositionof claim 103, wherein said antibody is a monoclonal antibody.
 105. Thecomposition of claim 104, wherein said antibody is anti-prominin-1 oranti-CD133 antibody.
 106. A recombinant CdtA polypeptide operably linkedto a ligand that binds specifically to an antigen expressed on thesurface of a cancerous epithelial cell type, wherein said recombinantCdtA polypeptide comprises at least one of the mutations C149A andC178A.
 107. A chimeric CdtA polypeptide comprising at least one of themutations C149A and C178A.
 108. An isolated nucleic acid sequenceencoding (i) a CdtA polypeptide comprising at least one of the mutationsC149A and C178A or (ii) a nucleic acid sequence that is at least 85%identical to the sequence of (i).
 109. A method for inhibiting theproliferation of a cancerous epithelial cell type comprising:administering a recombinant CdtA polypeptide according to claim 106.110.-119. (canceled)
 120. A method for inhibiting the proliferation of acancerous epithelial cell comprising: contacting said cell with arecombinant CdtA polypeptide according to claim
 106. 121. A method fortreating cancer comprising: administering a recombinant CdtA polypeptideaccording to claim
 106. 122. (canceled)
 123. (canceled)